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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 with small amounts of mRNA from the trapped loci frequently have monosomy of chromosome 5. A) Quantitative RT-PCR of P207 and P26. Expression of Ssbp3 mRNA in clone P207 was 1.4 ± 0.7% (n=3) by 3′ primers [P207(3′)] and 0.2 ± 0.01% (n=3) by flanking primers [P207(Fl)], compared with the parental PC12. The expression of P26 was 1.6 ± 0.3% (n=3). Data represent averages ± sem. B) Karyotype analysis of parental PC12 (PC12-mCAT1 and PC12-VAChT). Markers correspond to structurally abnormal chromosomes. C) Frequency of monosomy in each chromosome of PC12-mCAT1 and PC12-VAChT. Results are expressed as percentage of cells with monosomy at the indicated chromosome number. Five cells were counted from each parental cell line.
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Figure 7: Trapped clones with small amounts of mRNA from the trapped loci frequently have monosomy of chromosome 5. A) Quantitative RT-PCR of P207 and P26. Expression of Ssbp3 mRNA in clone P207 was 1.4 ± 0.7% (n=3) by 3′ primers [P207(3′)] and 0.2 ± 0.01% (n=3) by flanking primers [P207(Fl)], compared with the parental PC12. The expression of P26 was 1.6 ± 0.3% (n=3). Data represent averages ± sem. B) Karyotype analysis of parental PC12 (PC12-mCAT1 and PC12-VAChT). Markers correspond to structurally abnormal chromosomes. C) Frequency of monosomy in each chromosome of PC12-mCAT1 and PC12-VAChT. Results are expressed as percentage of cells with monosomy at the indicated chromosome number. Five cells were counted from each parental cell line.

Mentions: In contrast, in clone P207, we could hardly detect Ssbp3 mRNA (Fig. 7A, left bars). This result is consistent with the genomic Southern blot results showing that P207 had only one Ssbp3 allele. Using primers that flanked the insertion site (Fl primers in Fig. 4), we could hardly detect the PCR product (Fig. 7A, middle panel). Thus, the efficiency of gene trap was almost complete, which confirmed the result from the previous study (14).


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 with small amounts of mRNA from the trapped loci frequently have monosomy of chromosome 5. A) Quantitative RT-PCR of P207 and P26. Expression of Ssbp3 mRNA in clone P207 was 1.4 ± 0.7% (n=3) by 3′ primers [P207(3′)] and 0.2 ± 0.01% (n=3) by flanking primers [P207(Fl)], compared with the parental PC12. The expression of P26 was 1.6 ± 0.3% (n=3). Data represent averages ± sem. B) Karyotype analysis of parental PC12 (PC12-mCAT1 and PC12-VAChT). Markers correspond to structurally abnormal chromosomes. C) Frequency of monosomy in each chromosome of PC12-mCAT1 and PC12-VAChT. Results are expressed as percentage of cells with monosomy at the indicated chromosome number. Five cells were counted from each parental cell line.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Trapped clones with small amounts of mRNA from the trapped loci frequently have monosomy of chromosome 5. A) Quantitative RT-PCR of P207 and P26. Expression of Ssbp3 mRNA in clone P207 was 1.4 ± 0.7% (n=3) by 3′ primers [P207(3′)] and 0.2 ± 0.01% (n=3) by flanking primers [P207(Fl)], compared with the parental PC12. The expression of P26 was 1.6 ± 0.3% (n=3). Data represent averages ± sem. B) Karyotype analysis of parental PC12 (PC12-mCAT1 and PC12-VAChT). Markers correspond to structurally abnormal chromosomes. C) Frequency of monosomy in each chromosome of PC12-mCAT1 and PC12-VAChT. Results are expressed as percentage of cells with monosomy at the indicated chromosome number. Five cells were counted from each parental cell line.
Mentions: In contrast, in clone P207, we could hardly detect Ssbp3 mRNA (Fig. 7A, left bars). This result is consistent with the genomic Southern blot results showing that P207 had only one Ssbp3 allele. Using primers that flanked the insertion site (Fl primers in Fig. 4), we could hardly detect the PCR product (Fig. 7A, middle panel). Thus, the efficiency of gene trap was almost complete, which confirmed the result from the previous study (14).

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