title: Treatment of buffalo (Bubalus bubalis) donor cells with trichostatin A and 5-aza-2'-deoxycytidine alters their growth characteristics, gene expression and epigenetic status and improves the in vitro developmental competence, quality and epigenetic status of cloned embryos.
abstract: We examined the effects of treating buffalo skin fibroblast donor cells with trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, and 5-aza-2'-deoxycytidine (5azadC), a DNA methyltransferase (DNMT) inhibitor, on the cells and embryos produced by hand-made cloning. Treatment of donor cells with TSA or 5azadC resulted in altered expression levels of the HDAC1, DNMT1, DNMT3a, P53, CASPASE3 and CASPASE9 genes and global levels of acetylation of lysine at position 9 or 14 in histone 3 (H3K9/14ac), acetylation of lysine at position 5 in histone 4 (H4K5ac), acetylation of lysine at position 18 in histone 3 (H3K18ac) and tri-methylation of lysine at position 27 in histone 3 (H3K27me3). Moreover, global levels of DNA methylation and activity of DNMT1 and HDAC1 were decreased, while global acetylation of H3 and H3K9 was significantly increased in comparison to untreated cells. Simultaneous treatment of donor cells with TSA (50nM) and 5azadC (7.5nM) resulted in higher in vitro development to the blastocyst stage, reduction of the apoptotic index and the global level of H3K27 me3 and altered expression levels of HDAC1, P53, CASPASE3, CASPASE9 and DNMT3a in cloned blastocysts. Transfer of cloned embryos produced with donor cells treated with TSA led to the birth of a calf that survived for 21 days. These results show that treatment of buffalo donor cells with TSA and 5azadC improved developmental competence and quality of cloned embryos and altered their epigenetic status and gene expression, and that these beneficial effects were mediated by a reduction in DNA and histone methylation and an increase in histone acetylation in donor cells.
authors.affiliation: Animal Biotechnology Centre, National Dairy Research Institute, Karnal-132001, Haryana, India.
authors.forename: M
authors.initials: M
authors.lastname: Saini
authors.affiliation: Animal Biotechnology Centre, National Dairy Research Institute, Karnal-132001, Haryana, India.
authors.forename: N L
authors.initials: NL
authors.lastname: Selokar
authors.affiliation: Animal Biotechnology Centre, National Dairy Research Institute, Karnal-132001, Haryana, India.
authors.forename: H
authors.initials: H
authors.lastname: Agrawal
authors.affiliation: Animal Biotechnology Centre, National Dairy Research Institute, Karnal-132001, Haryana, India.
authors.forename: S K
authors.initials: SK
authors.lastname: Singla
authors.affiliation: Animal Biotechnology Centre, National Dairy Research Institute, Karnal-132001, Haryana, India.
authors.forename: M S
authors.initials: MS
authors.lastname: Chauhan
authors.affiliation: Animal Biotechnology Centre, National Dairy Research Institute, Karnal-132001, Haryana, India.
authors.forename: R S
authors.initials: RS
authors.lastname: Manik
authors.affiliation: Animal Biotechnology Centre, National Dairy Research Institute, Karnal-132001, Haryana, India.
authors.forename: P
authors.initials: P
authors.lastname: Palta
mesh_terms: D000107:Acetylation / Q000187:drug effects; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D001755:Blastocyst / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D002020:Buffaloes; D002478:Cells, Cultured; D019976:Cloning, Organism / Q000379:methods / Q000662:veterinary*; D019175:DNA Methylation / Q000187:drug effects; D015254:DNA Modification Methylases / Q000037:antagonists & inhibitors / Q000378:metabolism; D000077209:Decitabine; D004477:Ectogenesis / Q000187:drug effects*; D046149:Embryo Culture Techniques / Q000662:veterinary; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D044127:Epigenesis, Genetic / Q000187:drug effects*; D005260:Female; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects*; D056572:Histone Deacetylase Inhibitors / Q000494:pharmacology; D006657:Histones / Q000378:metabolism; D006877:Hydroxamic Acids / Q000494:pharmacology; D007194:India; D008745:Methylation / Q000187:drug effects; D011499:Protein Processing, Post-Translational / Q000187:drug effects
chemical_list: D004791:Enzyme Inhibitors; D056572:Histone Deacetylase Inhibitors; D006657:Histones; D006877:Hydroxamic Acids; C012589:trichostatin A; D000077209:Decitabine; D015254:DNA Modification Methylases; D001374:Azacitidine