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  1. Fulltext Cloning and Expression of Iranian Turkmen-thoroughbred Horse Follicle Stimulating Hormone in Pichia pastoris
    Elyasi Gorji Z, Amiri-Yekta A, Gourabi H, Hassani S, Fatemi N, Zerehdaran S, et al.
    Iran J Biotechnol, 2015 Jun;13(2):10-17.
    PMID: 28959285 DOI: 10.15171/ijb.1004
    BACKGROUND: Follicle stimulating hormone (FSH) plays an essential role in reproductive physiology and follicular development.

    OBJECTIVE: A new variant of the equine fsh (efsh) gene was cloned, sequenced, and expressed in Pichia pastoris (P. pastoris) GS115 yeast expression system.

    MATERIALS AND METHODS: The full-length cDNAs of the efshα and efshβ chains were amplified by reverse transcription polymerase chain reaction (RT-PCR) using the total RNA isolated from an Iranian Turkmen-thoroughbred horse's anterior pituitary gland. The amplified efsh chains were cloned into the pPIC9 vector and transferred into P. pastoris. The secretion of recombined eFSH using P. pastoris expression system was confirmed by Western blotting and immunoprecipitation (IP) methods.

    RESULTS: The DNA sequence of the efshβ chain accession number JX861871, predicted two putative differential nucleotide arrays, both of which are located in the 3'UTR. Western blotting showed a molecular mass of 13 and 18 kDa for eFSHα and eFSHβ subunits, respectively. The expression of desired protein was confirmed by protein G immunoprecipitation kit.

    CONCLUSIONS: eFSH successfully expressed in P. pastoris. These findings lay a foundation to improve ovulation and embryo recovery rates as well as the efficiency of total embryo-transfer process in mares.

  2. Improved in vitro development of cloned bovine embryos using S-adenosylhomocysteine, a non-toxic epigenetic modifying reagent
    Jafari S, Hosseini MS, Hajian M, Forouzanfar M, Jafarpour F, Abedi P, et al.
    Mol. Reprod. Dev., 2011 Aug;78(8):576-84.
    PMID: 21721066 DOI: 10.1002/mrd.21344
    In this study, fibroblast cells were stably transfected with mouse POU5F1 promoter-driven enhanced green fluorescent protein (EGFP) to investigate the effect of S-adenosylhomocysteine (SAH), the reversible non-toxic inhibitor of DNA-methyltransferases (DNMTs), at different intervals post-fusion on in vitro development of cloned bovine embryos. Treatment with SAH for 12 hr resulted in 54.6 ± 7.7% blastocyst production, which was significantly greater than in vitro fertilized embryos (IVF: 37.2 ± 2.7%), cloned embryos treated with SAH for 72 hr (31.0 ± 7.6%), and control cloned embryos (34.6 ± 3.6%). The fluorescence intensities of the EGFP-POU5F1 reporter gene at all intervals of SAH treatment, except of 72 hr, were significantly higher than control somatic cell nuclear transfers (SCNT) embryos. The intensity of DNA-methylation in cloned embryos treated with SAH for 48 hr was similar to that of IVF embryos, and was significantly lower than the other SCNT groups. The levels of H3K9 acetylation in all SCNT groups were significantly lower than IVF embryos. Real-time PCR analysis of gene expression revealed significantly higher expression of POU5F1 in cloned versus IVF blastocysts. Neither embryo production method (SCNT vs. IVF) nor the SAH treatment interval affected expression of the BCL2 gene. Cloned embryos at all intervals of SAH treatment, except for 24 hr, had significantly increased VEGF transcript compared to IVF and control SCNT embryos. It was suggested that the time interval of DNMT inhibition may have important consequences on different in vitro features of bovine SCNT, and the improving effects of DNMT inhibition on developmental competency of cloned embryos are restricted to a specific period of time preceding de novo methylation.
  3. Fulltext Epigenetic modification does not determine the time of POU5F1 transcription activation in cloned bovine embryos
    Jafari S, Hosseini SM, Hajian M, Forouzanfar M, Jafarpour F, Abedi P, et al.
    J Assist Reprod Genet, 2011 Nov;28(11):1119-27.
    PMID: 22020531 DOI: 10.1007/s10815-011-9638-1
    To investigate the effect of epigenetic modification on pattern, time and capacity of transcription activation of POU5F1, the key marker of pluripotency, in cloned bovine embryos.
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