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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

Southern blot analysis to confirm the integration of the gene trap vector and the integration sites in the genome. A) Genomic DNAs extracted from parental and mutant PC12 cells were digested with a restriction enzyme, ApaI. They were then probed with a puromycin probe (left panel) and an IRES probe (right panel). Bands are indicated by asterisks. Weak nonspecific bands (indicated by left arrowhead) were observed in all of the clones including the parental PC12. B) Genomic DNA from nonrevertant clones (A44, A28, A18, and A38) was purified before (Cre−), 1 d after (1D), and 3 d after (3D) the treatment Cre recombinase, digested by ApaI, and probed with a puromycin probe. Bands are indicated by white asterisks. C) Genomic DNAs extracted from the parental and mutant PC12 cells were digested with various restriction enzymes. A61 and P173 displayed two bands; P207 and P26 displayed one band. Genomic probes used are indicated in panel D. D) Genomic regions around the integration sites. Gene trap vectors are shown in red; probes in blue.
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Figure 5: Southern blot analysis to confirm the integration of the gene trap vector and the integration sites in the genome. A) Genomic DNAs extracted from parental and mutant PC12 cells were digested with a restriction enzyme, ApaI. They were then probed with a puromycin probe (left panel) and an IRES probe (right panel). Bands are indicated by asterisks. Weak nonspecific bands (indicated by left arrowhead) were observed in all of the clones including the parental PC12. B) Genomic DNA from nonrevertant clones (A44, A28, A18, and A38) was purified before (Cre−), 1 d after (1D), and 3 d after (3D) the treatment Cre recombinase, digested by ApaI, and probed with a puromycin probe. Bands are indicated by white asterisks. C) Genomic DNAs extracted from the parental and mutant PC12 cells were digested with various restriction enzymes. A61 and P173 displayed two bands; P207 and P26 displayed one band. Genomic probes used are indicated in panel D. D) Genomic regions around the integration sites. Gene trap vectors are shown in red; probes in blue.

Mentions: To examine the number of inserted vectors in the genome, we performed genomic Southern blot of these clones using probes within the gene trap vector (Fig. 5A). All clones had 1 vector/cell except P05, which appeared to have 2 bands by genomic Southern blot, and thus appeared to have 2 vectors/cell.


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)

Southern blot analysis to confirm the integration of the gene trap vector and the integration sites in the genome. A) Genomic DNAs extracted from parental and mutant PC12 cells were digested with a restriction enzyme, ApaI. They were then probed with a puromycin probe (left panel) and an IRES probe (right panel). Bands are indicated by asterisks. Weak nonspecific bands (indicated by left arrowhead) were observed in all of the clones including the parental PC12. B) Genomic DNA from nonrevertant clones (A44, A28, A18, and A38) was purified before (Cre−), 1 d after (1D), and 3 d after (3D) the treatment Cre recombinase, digested by ApaI, and probed with a puromycin probe. Bands are indicated by white asterisks. C) Genomic DNAs extracted from the parental and mutant PC12 cells were digested with various restriction enzymes. A61 and P173 displayed two bands; P207 and P26 displayed one band. Genomic probes used are indicated in panel D. D) Genomic regions around the integration sites. Gene trap vectors are shown in red; probes in blue.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Southern blot analysis to confirm the integration of the gene trap vector and the integration sites in the genome. A) Genomic DNAs extracted from parental and mutant PC12 cells were digested with a restriction enzyme, ApaI. They were then probed with a puromycin probe (left panel) and an IRES probe (right panel). Bands are indicated by asterisks. Weak nonspecific bands (indicated by left arrowhead) were observed in all of the clones including the parental PC12. B) Genomic DNA from nonrevertant clones (A44, A28, A18, and A38) was purified before (Cre−), 1 d after (1D), and 3 d after (3D) the treatment Cre recombinase, digested by ApaI, and probed with a puromycin probe. Bands are indicated by white asterisks. C) Genomic DNAs extracted from the parental and mutant PC12 cells were digested with various restriction enzymes. A61 and P173 displayed two bands; P207 and P26 displayed one band. Genomic probes used are indicated in panel D. D) Genomic regions around the integration sites. Gene trap vectors are shown in red; probes in blue.
Mentions: To examine the number of inserted vectors in the genome, we performed genomic Southern blot of these clones using probes within the gene trap vector (Fig. 5A). All clones had 1 vector/cell except P05, which appeared to have 2 bands by genomic Southern blot, and thus appeared to have 2 vectors/cell.

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