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The Expression, Locatization and Function of Nlrp4e in Preimplation Mouse Embryo

Author: ChangBo
Tutor: ZhangYong
School: Northwest University of Science and Technology
Course: Developmental Biology
Keywords: NLRP RNAi electroporation scanning confocal microscopy parthenogenetic activation
CLC: Q75
Type: Master's thesis
Year: 2012
Downloads: 36
Quote: 0
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Abstract


Increasing evidence suggests that some Nlrp genes are crucial for oogenesis,folliculogenesis and early embryonic development. Nlrp4e is one of seven copies of Nlrp4,which plays a putative role in the reproduction system in mice. Gene duplication is regardedas an important driving force behind the evolution of novel genes with new or alteredfunctions.We investigated the role of Nlrp4e in oocyte and preimplantation embryos bydetermining its expression profile and protein localization using quantitative real-timepolymerase chain reaction, immunoblotting, immunocytochemistry and immunofluorescence.Both Nlrp4e mRNA and protein accumulated during oogenesis. Moreover, Nlrp4e transcriptswere upregulated during the2-cell stage, which represents a crucial time for major embryonicgenome activation in the mouse. Transcripts then declined sharply and became almostundetectable, although the protein remained present through to the blastocyst stage.we develop a detailed electroporation method of efficient delivery of siRNA into mousepreimplantation embryos. Conditions for the electroporation, including the voltage, pulseduration, pulse number, electroporation buffer and an important step to weaken the zonapellucida, have been optimized by the introduction of Cy3labeled negative control smallinterfering RNA (siRNA) into mouse preimplantation embryos. Embryonic survival rate,transfection rate and blastocyst development rate were evaluated under the convertedfluorescence microscope, by embryos counting and statistical analysis. The best transfectionwas achieved in opti-MEM under the conditions of30V,1ms,3pulses, and the duration ofdigestion in tyrode’s solution was10s.Knockdown of Nlrp4e in fertilized eggs using RNA interference resulted in arresteddevelopment between the2-and8-cell stages in a dose-dependent manner. However, targetedinhibition of Nlrp4e in germinal-vesicle-stage oocytes had no phenotypic effects on oocytematuration. The above experiments were also carried out in parthenogenetic embryos todetermine the effects of Nlrp4e in embryos without a paternal genome. The results of thisstudy indicate that Nlrp4e, a maternal-zygotic-effect gene, may not be involved in oocytematuration but may play a critical role in early embryogenesis.

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