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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

Trapped clones expressing detectable amounts of mRNA exhibit the phenotype by their reduced amount of mRNA rather than expression of truncated products. A) Quantitative RT-PCR of A61 and P173. Expression of Btbd9 mRNA in clone A61 was 70.7 ± 4.5% (n=3) by 5′ primers [A61(5′)] and 34.7 ± 4.8% (n=3) by 3′ primers [A61(3′)], compared with the parental PC12 clones. Expression of Crlf3 mRNA in clone P173 was 54.0 ± 11.0% (n=2). Data represent averages ± sem. B) PCR from exons 1, 2, and 6 to IRES in the gene trap vector. PCR products were not detectable between exons 1/2 and IRES, while PCR product between exon 6 and IRES was clearly detected (an arrow). C) Knockdown of Btbd9 and Crlf3 in parental PC12 clones (middle panels) displayed reduction in the staining of synaptophysin similar to A61 (top right panel), and that of VAChT-EGFP similar to P173 (bottom right panel), respectively. D) Overexpression of truncated Btbd9 (tr-Btbd9) and Crlf3 (tr-Crlf3) does not change the phenotype of parental PC12. Transfected cells (arrows) and nontransfected cells (arrowheads) displayed similar staining of synaptophysin (left panels) and VAChT-EGFP (right panels), respectively. Cherry staining was used as a marker for transfected cells. Scale bars = 100 μm.
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Figure 6: Trapped clones expressing detectable amounts of mRNA exhibit the phenotype by their reduced amount of mRNA rather than expression of truncated products. A) Quantitative RT-PCR of A61 and P173. Expression of Btbd9 mRNA in clone A61 was 70.7 ± 4.5% (n=3) by 5′ primers [A61(5′)] and 34.7 ± 4.8% (n=3) by 3′ primers [A61(3′)], compared with the parental PC12 clones. Expression of Crlf3 mRNA in clone P173 was 54.0 ± 11.0% (n=2). Data represent averages ± sem. B) PCR from exons 1, 2, and 6 to IRES in the gene trap vector. PCR products were not detectable between exons 1/2 and IRES, while PCR product between exon 6 and IRES was clearly detected (an arrow). C) Knockdown of Btbd9 and Crlf3 in parental PC12 clones (middle panels) displayed reduction in the staining of synaptophysin similar to A61 (top right panel), and that of VAChT-EGFP similar to P173 (bottom right panel), respectively. D) Overexpression of truncated Btbd9 (tr-Btbd9) and Crlf3 (tr-Crlf3) does not change the phenotype of parental PC12. Transfected cells (arrows) and nontransfected cells (arrowheads) displayed similar staining of synaptophysin (left panels) and VAChT-EGFP (right panels), respectively. Cherry staining was used as a marker for transfected cells. Scale bars = 100 μm.

Mentions: To determine the amount of mRNA from the trapped genes, we used real-time PCR. In two cell lines (A61 and P173), approximately half of the mRNA from the trapped genes remained (Fig. 6A). When we measure the amount of mRNA of A61, we used primers and probes for real-time PCR at both upstream (5′ primers in Fig. 4) and downstream of the inserted site (3′ primers in Fig. 4) and found a reduction of mRNA by both primers and probes (Fig. 6A). We could not find splicing from the upstream exons (exons 1 and 2) to the splice acceptor of the gene trap vector (Fig. 6B), which suggests that the reduced amount of mRNA in the upstream of the inserted site was not because of the skipping of the upstream probe located at exons 3 and 4.


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)

Trapped clones expressing detectable amounts of mRNA exhibit the phenotype by their reduced amount of mRNA rather than expression of truncated products. A) Quantitative RT-PCR of A61 and P173. Expression of Btbd9 mRNA in clone A61 was 70.7 ± 4.5% (n=3) by 5′ primers [A61(5′)] and 34.7 ± 4.8% (n=3) by 3′ primers [A61(3′)], compared with the parental PC12 clones. Expression of Crlf3 mRNA in clone P173 was 54.0 ± 11.0% (n=2). Data represent averages ± sem. B) PCR from exons 1, 2, and 6 to IRES in the gene trap vector. PCR products were not detectable between exons 1/2 and IRES, while PCR product between exon 6 and IRES was clearly detected (an arrow). C) Knockdown of Btbd9 and Crlf3 in parental PC12 clones (middle panels) displayed reduction in the staining of synaptophysin similar to A61 (top right panel), and that of VAChT-EGFP similar to P173 (bottom right panel), respectively. D) Overexpression of truncated Btbd9 (tr-Btbd9) and Crlf3 (tr-Crlf3) does not change the phenotype of parental PC12. Transfected cells (arrows) and nontransfected cells (arrowheads) displayed similar staining of synaptophysin (left panels) and VAChT-EGFP (right panels), respectively. Cherry staining was used as a marker for transfected cells. Scale bars = 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Trapped clones expressing detectable amounts of mRNA exhibit the phenotype by their reduced amount of mRNA rather than expression of truncated products. A) Quantitative RT-PCR of A61 and P173. Expression of Btbd9 mRNA in clone A61 was 70.7 ± 4.5% (n=3) by 5′ primers [A61(5′)] and 34.7 ± 4.8% (n=3) by 3′ primers [A61(3′)], compared with the parental PC12 clones. Expression of Crlf3 mRNA in clone P173 was 54.0 ± 11.0% (n=2). Data represent averages ± sem. B) PCR from exons 1, 2, and 6 to IRES in the gene trap vector. PCR products were not detectable between exons 1/2 and IRES, while PCR product between exon 6 and IRES was clearly detected (an arrow). C) Knockdown of Btbd9 and Crlf3 in parental PC12 clones (middle panels) displayed reduction in the staining of synaptophysin similar to A61 (top right panel), and that of VAChT-EGFP similar to P173 (bottom right panel), respectively. D) Overexpression of truncated Btbd9 (tr-Btbd9) and Crlf3 (tr-Crlf3) does not change the phenotype of parental PC12. Transfected cells (arrows) and nontransfected cells (arrowheads) displayed similar staining of synaptophysin (left panels) and VAChT-EGFP (right panels), respectively. Cherry staining was used as a marker for transfected cells. Scale bars = 100 μm.
Mentions: To determine the amount of mRNA from the trapped genes, we used real-time PCR. In two cell lines (A61 and P173), approximately half of the mRNA from the trapped genes remained (Fig. 6A). When we measure the amount of mRNA of A61, we used primers and probes for real-time PCR at both upstream (5′ primers in Fig. 4) and downstream of the inserted site (3′ primers in Fig. 4) and found a reduction of mRNA by both primers and probes (Fig. 6A). We could not find splicing from the upstream exons (exons 1 and 2) to the splice acceptor of the gene trap vector (Fig. 6B), which suggests that the reduced amount of mRNA in the upstream of the inserted site was not because of the skipping of the upstream probe located at exons 3 and 4.

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