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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

Schematic representation of the gene trap retrovirus and the method for morphology-based screening. The gene trap retrovirus contains a SA and IRES upstream of the puromycin resistance and β-geo genes. Productive integration occurs in an intron of an expressed gene downstream of coding exons such that the puromycin resistance gene is spliced into the gene transcript. After selection with G418 and puromycin, the mutant clones were morphologically examined under a fluorescent microscope. The abnormal phenotype should be reverted to normal through Cre-mediated excision if the phenotype is due to the provirus insertion.
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Figure 1: Schematic representation of the gene trap retrovirus and the method for morphology-based screening. The gene trap retrovirus contains a SA and IRES upstream of the puromycin resistance and β-geo genes. Productive integration occurs in an intron of an expressed gene downstream of coding exons such that the puromycin resistance gene is spliced into the gene transcript. After selection with G418 and puromycin, the mutant clones were morphologically examined under a fluorescent microscope. The abnormal phenotype should be reverted to normal through Cre-mediated excision if the phenotype is due to the provirus insertion.

Mentions: As a second method to select clones, we used a PC12 cell line stably expressing VAChT fused with EGFP (VAChT-EGFP). We used VAChT as a marker of SLMVs, and in parental PC12 cells, it is predominantly targeted to SLMVs and clustered at the nerve terminals (5, 6). After retroviral infection, we performed visual screening under the fluorescent microscope to select clones that showed defects in the amount or distribution of VAChT-EGFP. We called this method morphology-based screening (Fig. 1).


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)

Schematic representation of the gene trap retrovirus and the method for morphology-based screening. The gene trap retrovirus contains a SA and IRES upstream of the puromycin resistance and β-geo genes. Productive integration occurs in an intron of an expressed gene downstream of coding exons such that the puromycin resistance gene is spliced into the gene transcript. After selection with G418 and puromycin, the mutant clones were morphologically examined under a fluorescent microscope. The abnormal phenotype should be reverted to normal through Cre-mediated excision if the phenotype is due to the provirus insertion.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Schematic representation of the gene trap retrovirus and the method for morphology-based screening. The gene trap retrovirus contains a SA and IRES upstream of the puromycin resistance and β-geo genes. Productive integration occurs in an intron of an expressed gene downstream of coding exons such that the puromycin resistance gene is spliced into the gene transcript. After selection with G418 and puromycin, the mutant clones were morphologically examined under a fluorescent microscope. The abnormal phenotype should be reverted to normal through Cre-mediated excision if the phenotype is due to the provirus insertion.
Mentions: As a second method to select clones, we used a PC12 cell line stably expressing VAChT fused with EGFP (VAChT-EGFP). We used VAChT as a marker of SLMVs, and in parental PC12 cells, it is predominantly targeted to SLMVs and clustered at the nerve terminals (5, 6). After retroviral infection, we performed visual screening under the fluorescent microscope to select clones that showed defects in the amount or distribution of VAChT-EGFP. We called this method morphology-based screening (Fig. 1).

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