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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

Identification of the proviral integration sites of clones A61, P173, P207, and P26. Insertion sites in Btbd9, Crlf3, Ssbp3, and Btf3l4 genes are illustrated by red triangles. Exons are represented as vertical black bars with the exon number indicated above. Primers used for quantitative RT-PCR are indicated by blue arrows. See Table 4 for nucleotide sequences at the junction of the endogenous gene and vector DNA.
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Figure 4: Identification of the proviral integration sites of clones A61, P173, P207, and P26. Insertion sites in Btbd9, Crlf3, Ssbp3, and Btf3l4 genes are illustrated by red triangles. Exons are represented as vertical black bars with the exon number indicated above. Primers used for quantitative RT-PCR are indicated by blue arrows. See Table 4 for nucleotide sequences at the junction of the endogenous gene and vector DNA.

Mentions: We tested whether the phenotypes could be reverted through the deletion of the viral insertion using Cre recombinase (Fig. 3B, right panels). We confirmed that the phenotypes of 14 of the 24 clones were reverted on infection with the Cre-encoding adenovirus (Table 2). The cells successfully reverted to the normal status were used to identify the inactivated genes. We attempted the 5′RACE method first because the mRNA upstream of the inserted site was expected to be trapped by the splice acceptor localized at the beginning of the gene trap vector. We also performed inverse PCR and SPLK PCR to identify the insertion sites. Table 3 shows the genes trapped using the retrovirus vectors and the methods used to identify the genes. Among these mutated clones, two clones were selected for further analysis (Figs. 3 and 4 and Table 4). Clone P173 displayed weak VAChT-EGFP fluorescence in the cell body and neurites. The trapped gene, Crlf3, is a cytokine receptor-like factor, and at present, its association with biogenesis or VAChT trafficking is unknown. Clone P207 had multibranched neurites and intense VAChT-EGFP fluorescence at the tip of the neurites. The responsible gene, Ssbp3 (also known as Ssdp1), has been reported to regulate head morphogenesis in mice (22).


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)

Identification of the proviral integration sites of clones A61, P173, P207, and P26. Insertion sites in Btbd9, Crlf3, Ssbp3, and Btf3l4 genes are illustrated by red triangles. Exons are represented as vertical black bars with the exon number indicated above. Primers used for quantitative RT-PCR are indicated by blue arrows. See Table 4 for nucleotide sequences at the junction of the endogenous gene and vector DNA.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Identification of the proviral integration sites of clones A61, P173, P207, and P26. Insertion sites in Btbd9, Crlf3, Ssbp3, and Btf3l4 genes are illustrated by red triangles. Exons are represented as vertical black bars with the exon number indicated above. Primers used for quantitative RT-PCR are indicated by blue arrows. See Table 4 for nucleotide sequences at the junction of the endogenous gene and vector DNA.
Mentions: We tested whether the phenotypes could be reverted through the deletion of the viral insertion using Cre recombinase (Fig. 3B, right panels). We confirmed that the phenotypes of 14 of the 24 clones were reverted on infection with the Cre-encoding adenovirus (Table 2). The cells successfully reverted to the normal status were used to identify the inactivated genes. We attempted the 5′RACE method first because the mRNA upstream of the inserted site was expected to be trapped by the splice acceptor localized at the beginning of the gene trap vector. We also performed inverse PCR and SPLK PCR to identify the insertion sites. Table 3 shows the genes trapped using the retrovirus vectors and the methods used to identify the genes. Among these mutated clones, two clones were selected for further analysis (Figs. 3 and 4 and Table 4). Clone P173 displayed weak VAChT-EGFP fluorescence in the cell body and neurites. The trapped gene, Crlf3, is a cytokine receptor-like factor, and at present, its association with biogenesis or VAChT trafficking is unknown. Clone P207 had multibranched neurites and intense VAChT-EGFP fluorescence at the tip of the neurites. The responsible gene, Ssbp3 (also known as Ssdp1), has been reported to regulate head morphogenesis in mice (22).

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