Promoter assay of Granzyme g in mouse 2-cell stage embryonic development
Date Issued
2014
Date
2014
Author(s)
Ou-Yang, Huan
Abstract
Mouse Granzyme g (Gzmg) is an important protease and specificitly expressed at 2-cell stage of mouse embryo. Maternal zygotic transition (MZT) that control from maternal massage to synthesize zygotic gene products promote preimplantation embryo development in mouse 2-cell stage. It is demonstrated that zygotic RNA synthesize is blocked in Gzmg knock-down 2-cell embryo. These data indicated that Gzmg is a necessary protein in MZT. Thus, we want to know what mechanisms regulate Gzmg specific expression in mouse embryo 2-cell stage. We cloned full-length Gzmg promoter from mouse genomic DNA, FL-pGzmg (-1696 ~ +28nt), and generated four deletion constructs of Gzmg promoter, Δ1- (-1369 ~ +28nt), Δ2- (-939 ~ +28nt), Δ3- (-711 ~ +28nt) and Δ4-pGzmg (-417 ~ +28nt). We replaced CMV promoter of pEGFP-N1 with Gzmg promoter. Furthermore, we co-microinjected different types of Gzmg promoter constructs and pCMV-IRES2-mCherry vector into zygote pronuclear, 2-cell nuclear and also added an aphidicolin drug to arrest the embryo cell cycle in S phase. We predicted transcription factor binding sites in promoter sequence by JASPAR and expression atlas website. The immunofluorescence method can observe transcription factors location in mouse preimplantation embryo. We found the highest EGFP expression Δ4-pGzmg-EGFP, but lower expression level in FL and Δ1-pGzmg-EGFP in both zygote pronuclear and 2-cell nuclear injection. This result demonstrated that time-specific transcriptional factors up-regulated Gzmg transcription activity by binding cis-element in -417~+28nt of Gzmg promoter sequence. In addition, The JASER database and expression atlas website revealed that Gabpa and Stat3 were predicted that may up-regulate Gzmg in 2-cell stage. Futhermore, Gabpa and Stat3 located in zygote and 2-cell nuclear by immunofluorescence. These data supposed that Gabpa and Stat3 may had transcriptional activity of regulatory genes in zygote and 2-cell stage. Surprisingly, we observed that Gabpa and Stat3 co-localized with Δ4-pGzmg-EGFP. -939~+28nt of Gzmg promoter sequence had cis-element that controled Gzmg transcription activity, and Gabpa and Stat3 may up-regulate Gzmg in 2-cell stage. Thus, Gabpa and Stat3 are maternal proteins and may up-regulate Gzmg to promote MZT.
Subjects
Granzme g
母源性胚源性基因轉變
啟動子分析
著床前胚胎
Type
thesis
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