The Expression Of H1foo Gene In Mouse Oocytes And Construction Of Tetracycline-inducible Expression System Of H1foo Gene

As a special subtype of linker histone, Linker histone H1foo plays an important roleduring oocytes maturation. To interfere the expression of H1foo at GV stage can induce cellcycle arrest, then lower the rate of maturation. What’s more, in the process of nucleartransfer, the somatic type linker histone H1can be rapidly replaced by the oocyte-specificlinker histone H1foo after the donor nucleus was transferred into the enucleated oocyte. Up tonow, such replacement phenomenon has been observed in the nuclear transfer processes ofdifferent species (e.g. Xenopus, mice and bovine), However, the mechanism of thereplacement is still unknown. The rapid replacement may be critical for the process of nuclearreprogramming.In order to further investigate the function of linker histone H1foo during oocytematuration, our experiments successfully constructed the eukaryotic expression vectorpH1foo-Venus, then we analyzed the rate of oocytes maturation and the morphology of thespindle after overexpression of H1foo. To explore the molecular mechanism of the promotionof oocyte maturation by H1foo, we mutated the potential target site(T-298) on H1foo by MPF;In addition, a tetracycline-inducible eukaryotic gene expression vector for H1foo was also beconstructed combined with Tet-On inducible expression system. Using the inducibleeukaryotic gene expression vector, the expression of linker histone H1foo can betimely-regulated. The results are as follows:1. In this study, we successfully obtained the full-length CDS sequence of H1foo frommouse ovarian tissue by RT-PCR.. the result of Fluorescence microscopy showed thateukaryotic expression vector pH1foo-Venus correctly expressed and localized in the cells.Using pH1foo-Venus and pVenus vectors, we obtained their mRNAs by in vitro transcription,then after microinjection, the fluorescence protein expression of the H1foo-Venus could beobserved in oocyte nucleus. After overexpression of H1foo-Venus and Venus, the rate ofmaturation in H1foo-Venus injected group (54.3%) was significantly higher than that ofVenus injected group (36.25%). The mutation of T-298on H1foo had no effect on its localization in the oocytes. What’s more, immunofluorescence staining showed thatoverexpression of H1foo had no effect on the spindle assembly, the spindle morphology wasnormal.2. On the basis of pH1foo-Venus, a tetracycline-inducible expression vector was alsosuccessfully constructed, and fluorescence microscopic images showed that the inducibleexpression vector could be induced by tetracycline at a particular time.The results suggested that: The eukaryotic expression vector for mouse H1foo wassuccessfully expressed and localized in mouse oocytes; the overexpression of H1foo inmouse oocytes can significantly promote the oocyte maturation; Overexpression of H1foo hasno effect on the formation of oocytes spindle; The mutation of T-298on H1foo had no effecton the localization of H1foo in mouse oocytes; The tetracycline-inducible expression vectorwas constructed successfully; The expression of the tetracycline-inducible expression vectorcan be regulated in somatic cells at a particular time; Our experiments laid a foundation forour further study on the mechanism of H1foo during the cell cycle of oocyte.