Mutagenesis by Retroviral Insertion in Chemical Mutagen-Generated Quasi-Haploid Mammalian Cells

Kim, Sung O.; Ha, Soon Duck; Lee, Sangun; Stanton, Sarah G.; Beutler, Bruce; Han, Jiahuai

doi:10.2144/000112390

Cited by 8 publications

(13 citation statements)

References 45 publications

(34 reference statements)

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“…Recent studies have shown that TNF-␣ surface delivery and secretion is regulated through a process involving cholesterol-dependent lipid raft formation at the phagocytic cup of activated macrophages (7,12); however, it is unknown how TNF-␣ surface delivery is regulated and whether it can be facilitated upon TLR activation. We used a functional gene identification procedure using retrovirus integration-generated mutagenesis (13)(14)(15) and identified that cathepsin B activity was required for optimal TNF-␣ transportation to the plasma membrane. Cathepsin B is a lysosomal cysteine protease involved in the degradation of cellular proteins in lysosomes.…”

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confidence: 99%

Cathepsin B Is Involved in the Trafficking of TNF-α-Containing Vesicles to the Plasma Membrane in Macrophages

Martins

Khazaie

et al. 2008

The Journal of Immunology

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113

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TNF-α is a potent proinflammatory cytokine, essential for initiating innate immune responses against invading microbes and a key mediator involved in the pathogenesis of acute and chronic inflammatory diseases. To identify molecules involved in the production of TNF-α, we used a functional gene identification method using retroviral integration-mediated mutagenesis, followed by LPS-stimulated TNF-α production analysis in macrophages. We found that cathepsin B, a lysosomal cysteine proteinase, was required for optimal posttranslational processing of TNF-α in response to the bacterial cell wall component LPS. Mouse bone marrow-derived macrophages from cathepsin B-deficient mice and macrophages treated with the cathepsin B-specific chemical inhibitor CA074 methyl ester or small interfering RNA against cathepsin B secreted significantly less TNF-α than wild-type or nontreated macrophages. We further showed that the inhibition of cathepsin B caused accumulation of 26-kDa pro-TNF-containing vesicles. Ectopic expression of GFP-conjugated pro-TNF further suggests that pro-TNF failed to reach the plasma membrane without intracellular cathepsin B activity. Altogether, these data suggest that intracellular cathepsin B activity is involved in the TNF-α-containing vesicle trafficking to the plasma membrane.

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confidence: 99%

Cathepsin B Is Involved in the Trafficking of TNF-α-Containing Vesicles to the Plasma Membrane in Macrophages

Martins

Khazaie

et al. 2008

The Journal of Immunology

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113

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“…As previously reported, a combination of chemical and insertional mutagenesis can be used for the mutagenic screen in cultured mammalian cells (Kim et al, 2007). We generated a pool of theoretically quasi-haploid RAW 264.7 cells, which have 99.5% of all genes in random mutation after eight rounds of N-ethyl-N-nitrosourea (ENU) treatment (Kim et al, 2007).…”

Section: Resultsmentioning

confidence: 99%

“…We generated a pool of theoretically quasi-haploid RAW 264.7 cells, which have 99.5% of all genes in random mutation after eight rounds of N-ethyl-N-nitrosourea (ENU) treatment (Kim et al, 2007). Then, the retroviral insertion was introduced into the quasi-haploid cell pool to abolish gene expression or produce a truncated gene product.…”

Section: Resultsmentioning

confidence: 99%

“…Although retroviral insertional mutagenesis may be a powerful tool, the diploidity in eukaryotic cells is an obstacle yet to be solved. To overcome this problem, chemical mutagenesis could be applied in addition to retroviral insertional mutagenesis (Kim et al, 2007). In principle, chemical mutagenesis in vitro could allow a high rate of mutations by multiple applications and generate a pool of cells that can be regarded as 'quasi-haploid.'…”

Section: Introductionmentioning

confidence: 99%

“…In principle, chemical mutagenesis in vitro could allow a high rate of mutations by multiple applications and generate a pool of cells that can be regarded as 'quasi-haploid.' Then, the introduction of a retrovirus into the cells may make randomly disrupted genes that have been rendered haploid, permitting a rapid identification of each mutation that causes a phenotype of interest (Kim et al, 2007). We adapted the gene disruption system using chemical and retroviral insertional mutation in order to understand the molecular mechanism underlying LeTx-induced macrophage death.…”

Section: Introductionmentioning

confidence: 99%

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AMPD3 is involved in anthrax LeTx-induced macrophage cell death

Lee¹,

Wang²,

Kim³

et al. 2011

Protein Cell

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The responses of macrophages to Bacillus anthracis infection are important for the survival of the host, since macrophages are required for the germination of B. anthracis spores in lymph nodes, and macrophage death exacerbates anthrax lethal toxin (LeTx)-induced organ collapse. To elucidate the mechanism of macrophage cell death induced by LeTx, we performed a genetic screen to search for genes associated with LeTx-induced macrophage cell death. RAW 264.7 cells, a macrophage-like cell line sensitive to LeTx-induced death, were randomly mutated and LeTx-resistant mutant clones were selected. AMP deaminase 3 (AMPD3), an enzyme that converts AMP to IMP, was identified to be mutated in one of the resistant clones. The requirement of AMPD3 in LeTxinduced cell death of RAW 264.7 cells was confirmed by the restoration of LeTx sensitivity with ectopic reconstitution of AMPD3 expression. AMPD3 deficiency does not affect LeTx entering cells and the cleavage of mitogen-activated protein kinase kinase (MKK) by lethal factor inside cells, but does impair an unknown downstream event that is linked to cell death. Our data provides new information regarding LeTx-induced macrophage death and suggests that there is a key regulatory site downstream of or parallel to MKK cleavage that controls the cell death in LeTx-treated macrophages.

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Phenotype Based Functional Gene Screening Using Retrovirus-Mediated Gene Trapping in Quasi-Haploid RAW 264.7 Cells

Kim

2010

Methods in Molecular Biology

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In vitro random mutagenesis, followed by phenotype screening, provides a rapid and convenient tool for identifying novel genes involved in the phenotype of interest. However, the forward mutagenic approach in mammalian somatic cells is seriously limited by the diploidic nature of the genome. To overcome this impediment, we developed a method that allows functional screening for both haploid insufficient and sufficient genes involved in the phenotype of interest, utilizing a retrovirus gene trap mutagenesis in chemical mutagen-generated quasi-haploid cells. This method was used to identify novel host genes that are required for macrophage sensitivity to anthrax lethal toxin.

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Mutagenesis by Retroviral Insertion in Chemical Mutagen-Generated Quasi-Haploid Mammalian Cells

Cited by 8 publications

References 45 publications

Cathepsin B Is Involved in the Trafficking of TNF-α-Containing Vesicles to the Plasma Membrane in Macrophages

Cathepsin B Is Involved in the Trafficking of TNF-α-Containing Vesicles to the Plasma Membrane in Macrophages

AMPD3 is involved in anthrax LeTx-induced macrophage cell death

Phenotype Based Functional Gene Screening Using Retrovirus-Mediated Gene Trapping in Quasi-Haploid RAW 264.7 Cells

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