O-12: Study of Expression of DevelopmentalGenes in SCNT Cloned Embryos

(SCNT) embryos of buffaloes. 2. To study gene expression profile of important developmental genes at different stages of SCNT cloned embryo. 3. To study epigenetic reprogramming during early developments of SCNT embryos Materials and Methods: Expression analysis of developmental genes was done in different (ovarian granulose and cumulus and skin fibroblasts) donor cells; in vitro maturing oocytes and different stages of developing SCNT cloned embryos. IVF embryos were kept as controls. Effect of various media, hormones and other supplements at different levels was studied on SCNT embryos production rates and their related gene expression profile. Cultures were developed from different donor tissues and passaged serially. Primary oocytes from abattoir buffalo ovaries were matured in vitro. IVM oocytes were enucleated using micro manipulators and one cell was transferred as nuclear donor. Cell-oocyte complex was stimulated using two DC pulses using ECM-2001 and chemically activated by using cycloheximde and then cultured in TCM-199. The c-DNA was prepared from donor cells, oocytes and developing embryos (SCNT and IVF) using a cell to cDNA kit (Ambion). Real-time PCR primers were designed using BEACON DESIGNER and Q-PCR was performed on Mx3000p (Stratagene) using SYBR Green supermix. Data on mRNA expression wereanalysed using light cycler software to determine differences in gene expression pattern. Results: Expression of chromatin remodelling mRNAs; HDAC1, DNMT1, DNMT3a and DNMT3b was checked at various passages of skin fibroblast, cumulus and granulosa cell lines. HDAC1 mRNA expression was significantly lower in cumulus cells than skin fibroblast and granulosa cells. DNMT1 mRNA expression was significantlyhigher in cumulus cells than other cell types. DNMT3a expression was lower in granulosa cells than other cells. DNMT3b expression was higher in cumulus cells. Thus cumulus cells showed better expression of remodelling genes than other donor cells. The mRNA expressions of GAPDH (reference), Cx43, GDF-9, FGF-4 and Fibronectin were studied to evaluate effects of epigenetic modification by lectin supplementation during oocyte maturation. Relative mRNA abundance of all the four genes was significantly higher in 10 μg/ ml lectin dose in comparison to other treatment groups, showing utility of lectins in IVM of oocytes. IGF-1 and IGF-2 mRNA transcript level was significantly higher in NT embryos developed from different cell types than IVF embryos. IGF-1R and IGF-2R mRNA transcript level from cumulus cells was comparable to IVF. The embryo production rate of SCNT buffalo from granulosa and skin fibroblasts was significantly lower than from cumulus cells and IVF.Conclusion: Our results indicate that cumulus cells are best nuclear donor for SCNT clone production because of their better reprogramming capabilities. The mRNA transcript levels of developmental genes were significantly altered by culture medium and media supplements. There is a possibility of improving gene expression and efficiency of SCNT cloned embryos production by epigenetic modification at different stages for developing ES cells from respective donor for therapeutic applications.