| 研究課題/領域番号 |
19K16020
|
|---|
| 研究機関 | 筑波大学 |
|---|
研究代表者 |
DINH T.H.TRA 筑波大学, 医学医療系, 研究員 (30816476)
|
|---|
| 研究期間 (年度) |
2019-04-01 – 2022-03-31
|
| キーワード | germ cell / primordial germ cell / embryogenesis / Cables2 / Smad2 / Nanog |
|---|
| 研究実績の概要 |
Primordial germ cell (PGC) development is one of the most important biological process in which Nanog and Smad2 contribute into the germ cell specification during gastrulation. Cables2 was recently found as a novel transcription cofactor of Nodal/Smad2 signaling pathways in regulating Nanog expression in mouse gastrulation. Furthermore, Cables2 is broadly expressed in mouse embryonic and adult tissues with the highest RNA level in testis, suggesting the existence and contribution of Cables2 in male germ cell lineage. Therefore, the temporal function of Cables2 in embryogenesis, specifically in germ cell development should be explored. Moreover, the Cables2-binding protein facilitating Cables2-Smad2/Nanog in primordial germ cells and germ cells will be discovered.
The conventional Cables2 knock-out (KO) embryos are arrested the normal development from E6.5. Therefore, the Cre/loxP system will be applied to analyze the PGC and germ cell-specific function of Cables2. Using the CRISPR/Cas9 system, the floxed Cables2 mouse with loxP sites flanking exon1 was successfully generated. The homozygous mice were propagated by intercrossing of heterozygous floxed Cables2 mice. Generally, the physical appearance was normal in both heterozygous and homozygous mice suggesting that Cables2 conditional mice are normal and healthy. The establishment of PGC-like cells from pluripotent stem cells is setting up. In additional, to observe and collect the stable PGC in vivo, the reporter mice of Cables2 were generated and be propagating.
|
| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
Up to date, there is no commercially valuable Cables2 antibody and expression of Cables2 in vivo remains unclear. Therefore, bicistronic Cables2 knock-in reporter mice that expressed Cables2 tagged with fluorescent reporter was generated. In this model, C-teminal of Cables2 was knocked in with 3xFLAG and 2A-mediated tdTomato. The preliminary data from screening analysis revealed the expression of Cables2 in mouse adult tissues, specially in ovary and testis.
|
| 今後の研究の推進方策 |
The homozygous floxed Cables2 mice will be mated with several Cre mouse strains which which plays essential roles in germ cell development. The conditional knock-out Cables2 will be crossed with Nanog-GFP reporter mouse and the embryos will be analyzed during the specification to differentiation of primordial germ cell and germ cell. Besides, the Cables2 reporter mice will be analyzed and characterized at early development and adult stage to reveal the expression and cellular localization of Cables2 protein. Moreover, the reporter model will be useful to investigate the Cables2-binding proteins in vivo and the mechanism of how Cables2 regulates Nanog expression and Smad2 transcriptional activation in PGCs and male germ cells will be discovered further.
|
| 次年度使用額が生じた理由 |
In this year, the gene-modified mice were generated and propagated overall. The incurring amount therefore will be saved and used next year to maintain and breeding mouse strains, purchasing reagents, antibodies and experimental kit for screening analysis.
|
