Enrichment of Insertional Mutants Following Retrovirus Gene Trap Selection

Chang, Wen; Hubbard, S C; Friedel, C; Ruley, H E

doi:10.1006/viro.1993.1182

Cited by 33 publications

(30 citation statements)

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“…2A). This frequency of selection (1 MGBG-resistant cell in 10 5 hygromycin-resistant cells) was similar to the frequency of selection of hemizygous genes in the study by Chang et al (15). The MGBG-resistant 432c cells also had a significant decrease in polyamine transport activity (Fig.…”

Section: Screening For Mgbg Resistance In Cho Cellssupporting

confidence: 85%

“…1), we incubated ϳ2 ϫ 10 7 cells with the U3Hygro retrovirus, from which 1 ϫ 10 5 hygromycinresistant CHO cell colonies were selected. Chang et al (15) found a similar frequency of hygromycin-resistant colonies using the same virus and CHO cells. After further selection with 10 M MGBG, a concentration that will kill all uninfected CHO cells in 4 -5 days (data not shown), a total of 38 colonies survived.…”

Section: Screening For Mgbg Resistance In Cho Cellsmentioning

confidence: 75%

“…Theoretically, the number of retroviral insertion events in this study was enough to cover the whole genome (15), and the hemizygous character of most CHO genes should facilitate a loss of gene function (26). However, under our experimental conditions, only one clone was isolated that was resistant to MGBG, and this clone was deficient in polyamine transport activity.…”

Section: Discussionmentioning

confidence: 90%

“…Viral gene insertion can disrupt critical controlling elements that can disrupt expression of genes. In theory, a library of 10 4 -10 5 hygromycin-resistant clones containing proviruses would cover all genes (15). In our gene trap experiment (Fig.…”

Section: Screening For Mgbg Resistance In Cho Cellsmentioning

confidence: 89%

“…The copy number of the U3 insertion in 432c cells was determined by Southern blot analysis using the neomycin resistance gene as a probe. U3Hygro harbors this gene as a marker for viral infection and insertion (15). Genomic DNA of 432c cells was digested with HindIII, SspI, and StuI, which do not cut U3Hygro, followed by electrophoresis and blotting.…”

Section: Screening For Mgbg Resistance In Cho Cellsmentioning

confidence: 99%

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TATA-binding Protein-associated Factor 7 Regulates Polyamine Transport Activity and Polyamine Analog-induced Apoptosis

Fukuchi

Hiipakka

Kokontis

et al. 2004

Journal of Biological Chemistry

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Identification of the polyamine transporter gene will be useful for modulating polyamine accumulation in cells and should be a good target for controlling cell proliferation. Polyamine transport activity in mammalian cells is critical for accumulation of the polyamine analog methylglyoxal bis(guanylhydrazone) (MGBG) that induces apoptosis, although a gene responsible for transport activity has not been identified. Using a retroviral gene trap screen, we generated MGBG-resistant Chinese hamster ovary (CHO) cells to identify genes involved in polyamine transport activity. One gene identified by the method encodes TATA-binding protein-associated factor 7 (TAF7), which functions not only as one of the TAFs, but also a coactivator for c-Jun. TAF7-deficient cells had decreased capacity for polyamine uptake (20% of CHO cells), decreased AP-1 activation, as well as resistance to MGBG-induced apoptosis. Stable expression of TAF7 in TAF7-deficient cells restored transport activity (55% of CHO cells), AP-1 gene transactivation (100% of CHO cells), and sensitivity to MGBG-induced apoptosis. Overexpression of TAF7 in CHO cells did not increase transport activity, suggesting that TAF7 may be involved in the maintenance of basal activity. c-Jun NH 2 -terminal kinase inhibitors blocked MGBG-induced apoptosis without alteration of polyamine transport. Decreased TAF7 expression, by RNA interference, in androgen-independent human prostate cancer LNCaP104-R1 cells resulted in lower polyamine transport activity (25% of control) and resistance to MGBG-induced growth arrest. Taken together, these results reveal a physiological function of TAF7 as a basal regulator for mammalian polyamine transport activity and MGBG-induced apoptosis.

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Section: Screening For Mgbg Resistance In Cho Cellssupporting

confidence: 85%

Section: Screening For Mgbg Resistance In Cho Cellsmentioning

confidence: 75%

Section: Discussionmentioning

confidence: 90%

Section: Screening For Mgbg Resistance In Cho Cellsmentioning

confidence: 89%

Section: Screening For Mgbg Resistance In Cho Cellsmentioning

confidence: 99%

See 3 more Smart Citations

TATA-binding Protein-associated Factor 7 Regulates Polyamine Transport Activity and Polyamine Analog-induced Apoptosis

Fukuchi

Hiipakka

Kokontis

et al. 2004

Journal of Biological Chemistry

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Isolation and Characterization of a Near-Haploid Human Cell Line

Kotecki

1999

Experimental Cell Research

140

134

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Gene Trap Mutagenesis

Abuin

Hansen

Zambrowicz

Conditional Mutagenesis: An Approach to Disease Models

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Our ability to genetically manipulate the mouse has had a great impact on medical research over the last few decades. Mouse genetics has developed into a powerful tool for dissecting the genetic causes of human disease and identifying potential targets for pharmaceutical intervention. With the recent sequencing of the human and mouse genomes, a large number of novel genes have been identified whose function in normal and disease physiology remains largely unknown. Government-sponsored multinational efforts are underway to analyze the function of all mouse genes through mutagenesis and phenotyping, making the mouse the interpreter of the human genome. A number of technologies are available for the generation of mutant mice, including gene targeting, gene trapping and transposon, chemical or radiation-induced mutagenesis. In this chapter, we review the current status of gene trapping technology, including its applicability to conditional mutagenesis.

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Enrichment of Insertional Mutants Following Retrovirus Gene Trap Selection

Cited by 33 publications

References 0 publications

TATA-binding Protein-associated Factor 7 Regulates Polyamine Transport Activity and Polyamine Analog-induced Apoptosis

TATA-binding Protein-associated Factor 7 Regulates Polyamine Transport Activity and Polyamine Analog-induced Apoptosis

Isolation and Characterization of a Near-Haploid Human Cell Line

Gene Trap Mutagenesis

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