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Uncovering genes required for neuronal morphology by morphology-based gene trap screening with a revertible retrovirus vector.

Hashimoto Y, Muramatsu K, Kunii M, Yoshimura S, Yamada M, Sato T, Ishida Y, Harada R, Harada A - FASEB J. (2012)

Bottom Line: The first gene was BTB/POZ domain-containing protein 9 (Btbd9), which is associated with restless legs syndrome.The second gene was cytokine receptor-like factor 3 (Crlf3), whose involvement in the nervous system remains unknown.The third gene was single-stranded DNA-binding protein 3 (Ssbp3), a gene known to regulate head morphogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan.

ABSTRACT
The molecular mechanisms of neuronal morphology and synaptic vesicle transport have been largely elusive, and only a few of the molecules involved in these processes have been identified. Here, we developed a novel morphology-based gene trap method, which is theoretically applicable to all cell lines, to easily and rapidly identify the responsible genes. Using this method, we selected several gene-trapped clones of rat pheochromocytoma PC12 cells, which displayed abnormal morphology and distribution of synaptic vesicle-like microvesicles (SLMVs). We identified several genes responsible for the phenotypes and analyzed three genes in more detail. The first gene was BTB/POZ domain-containing protein 9 (Btbd9), which is associated with restless legs syndrome. The second gene was cytokine receptor-like factor 3 (Crlf3), whose involvement in the nervous system remains unknown. The third gene was single-stranded DNA-binding protein 3 (Ssbp3), a gene known to regulate head morphogenesis. These results suggest that Btbd9, Crlf3, and Ssbp3 regulate neuronal morphology and the biogenesis/transport of synaptic vesicles. Because our novel morphology-based gene trap method is generally applicable, this method is promising for uncovering novel genes involved in the function of interest in any cell lines.

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Related in: MedlinePlus

Features of clone A61. A) Parental PC12-mCAT1 cells (left panel), A61 cells (middle panel), and A61 cells after Cre/loxP-mediated recombination (right panel) were stained using an antibody against synaptophysin. A61 displayed weak expression of synaptophysin, but this phenotype recovered after infection with the adenovirus-encoding Cre. B) A61 cells were resistant to aerolysin but became sensitive to aerolysin after Cre/loxP-mediated recombination. Scale bars = 100 μm.
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Figure 2: Features of clone A61. A) Parental PC12-mCAT1 cells (left panel), A61 cells (middle panel), and A61 cells after Cre/loxP-mediated recombination (right panel) were stained using an antibody against synaptophysin. A61 displayed weak expression of synaptophysin, but this phenotype recovered after infection with the adenovirus-encoding Cre. B) A61 cells were resistant to aerolysin but became sensitive to aerolysin after Cre/loxP-mediated recombination. Scale bars = 100 μm.

Mentions: One of these clones, A61, displayed a significant decrease of synaptophysin staining (Fig. 2A). We identified the trapped gene of this clone as Btbd9 (Table 1), a gene associated with restless legs syndrome (21).


Uncovering genes required for neuronal morphology by morphology-based gene trap screening with a revertible retrovirus vector.

Hashimoto Y, Muramatsu K, Kunii M, Yoshimura S, Yamada M, Sato T, Ishida Y, Harada R, Harada A - FASEB J. (2012)

Features of clone A61. A) Parental PC12-mCAT1 cells (left panel), A61 cells (middle panel), and A61 cells after Cre/loxP-mediated recombination (right panel) were stained using an antibody against synaptophysin. A61 displayed weak expression of synaptophysin, but this phenotype recovered after infection with the adenovirus-encoding Cre. B) A61 cells were resistant to aerolysin but became sensitive to aerolysin after Cre/loxP-mediated recombination. Scale bars = 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3475256&req=5

Figure 2: Features of clone A61. A) Parental PC12-mCAT1 cells (left panel), A61 cells (middle panel), and A61 cells after Cre/loxP-mediated recombination (right panel) were stained using an antibody against synaptophysin. A61 displayed weak expression of synaptophysin, but this phenotype recovered after infection with the adenovirus-encoding Cre. B) A61 cells were resistant to aerolysin but became sensitive to aerolysin after Cre/loxP-mediated recombination. Scale bars = 100 μm.
Mentions: One of these clones, A61, displayed a significant decrease of synaptophysin staining (Fig. 2A). We identified the trapped gene of this clone as Btbd9 (Table 1), a gene associated with restless legs syndrome (21).

Bottom Line: The first gene was BTB/POZ domain-containing protein 9 (Btbd9), which is associated with restless legs syndrome.The second gene was cytokine receptor-like factor 3 (Crlf3), whose involvement in the nervous system remains unknown.The third gene was single-stranded DNA-binding protein 3 (Ssbp3), a gene known to regulate head morphogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan.

ABSTRACT
The molecular mechanisms of neuronal morphology and synaptic vesicle transport have been largely elusive, and only a few of the molecules involved in these processes have been identified. Here, we developed a novel morphology-based gene trap method, which is theoretically applicable to all cell lines, to easily and rapidly identify the responsible genes. Using this method, we selected several gene-trapped clones of rat pheochromocytoma PC12 cells, which displayed abnormal morphology and distribution of synaptic vesicle-like microvesicles (SLMVs). We identified several genes responsible for the phenotypes and analyzed three genes in more detail. The first gene was BTB/POZ domain-containing protein 9 (Btbd9), which is associated with restless legs syndrome. The second gene was cytokine receptor-like factor 3 (Crlf3), whose involvement in the nervous system remains unknown. The third gene was single-stranded DNA-binding protein 3 (Ssbp3), a gene known to regulate head morphogenesis. These results suggest that Btbd9, Crlf3, and Ssbp3 regulate neuronal morphology and the biogenesis/transport of synaptic vesicles. Because our novel morphology-based gene trap method is generally applicable, this method is promising for uncovering novel genes involved in the function of interest in any cell lines.

Show MeSH
Related in: MedlinePlus