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Abstract Hlfoo(oocyte-specific linker histone H1) is a linker histone specifically expressed in oocytes and early embryos in mammalian, it plays a crucial role in oogenesis, fertilization and embryogenesis. The objectives of this study were to clone swine H1foo gene and construct a eukaryotic expression vector pVenus-H1foo. Firstly, we applied the 5'RACE and RT-PCR to obtain the complete CDS of swine H1foo gene, the sequence information was submitted to GenBanK (GenBank accession No.: HQ915640). The CDS of swine H1foo gene was linked into pMD19-T and confirmed by digestion of restriction enzyme, and then the H1foo CDS was cloned into pVenus to construct a eukaryotic expression vector pVenus-H1foo. After pVenus-H1foo transfected into Hela cells mediated by Lipofectamine 2000, it was efficiently expressed in hela cells and identified by observation of fluorescence microscopy and RT-PCR. In addition, after transcripted in vitro, the mRNA of H1foo-venus was microinjected into swine oocytes, the expression and location was identified under fluorescence microscopy. The result of sequence analysis showed that the CDS region of the swine H1foo gene included 1 041 nucleotides, encoding for 346 amino acids(Mol.wt.: 36.45kD), and the nucleotide similarity of H1foo between swine and bovine, human, mice was 75.7%, 67.9% and 54.3%,respectively. After transfection and microinjection, the H1foo was expressed and localized accurately both in Hela cell nucleus and swine oocyte nucleus. We had cloned swine H1foo gene and constructed the expression vector pVenus-H1fooin; The mRNA of H1foo-venus transcripted in vitro was expressed and localized accurately in swine oocytes, which greatly facilitates the further research of H1foo in oocytes maturation and nuclear transplantation.
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Received: 13 June 2011
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