Trichostatin A improved epigenetic modifications of transfected cells but did not improve subsequent cloned embryo development.
Reprogramming impairment of DNA methylation may be partly responsible for the low efficiency in somatic cell nuclear transfer. In this study, bovine fibroblast cells were transfected with enhancer green fluorescence protein (eGFP), and then treated with a histone-deacetylase inhibitor, trichostatin A (TSA). The results showed that the effect of TSA on transfected cells was dose dependent. When the TSA concentration was over 5 ng/ml, cell proliferation was significantly inhibited. The majority of the cells died when TSA reached 100 ng/ml (P < 0.01). The number of cells in the S phase was significantly decreased in the 5- to 50-ng/ml TSA-treated groups, while the majority of the cells were at the G0/G1 phases. The number of eGFP-expressed cells were approximately twofold higher in 25-ng/ml (30.5%) and 50-ng/ml (29.5%) TSA groups than the control (15.0%). Reduced DNA methylation and improved histone acetylation were observed when the cells were treated with 10 to 50 ng/ml of TSA. Transfer of the TSA-treated cells to enucleated recipient oocytes resulted in similar cleavage rates among the experimental groups and the control. Cells treated with 50 ng/ml of TSA resulted in significantly lower blastocyst development (9.9%) than the other experimental and the control groups (around 20%). Analysis of the putative blastocysts showed that 86.7% of the embryos derived from TSA-treated cells were eGFP positive, which was higher than that from untreated cells (68.8%). In conclusion, treatment of transfected cells with TSA decreased the genome DNA methylation level, increased histone acetylation, and eGFP gene expression was activated. Donor cells with reduced DNA methylation did not improve subsequent cloned embryo development; however, transgene expression was improved in cloned embryos.
The Key Laboratory of Mammalian Reproductive Biology and Biotechnology of the Ministry of Education, Inner Mongolia University, Hohhot, China.
Xia
X
Wu
Yan
Y
Li
Guang-Peng
GP
Li
Dongshan
D
Yang
Yongli
Y
Yue
Lingling
L
Wang
Kehan
K
Li
Penghui
P
Xin
Shorgan
S
Bou
Haiquan
H
Yu
D000107:Acetylation / Q000187:drug effects; D000818:Animals; D002417:Cattle / Q000196:embryology* / Q000235:genetics / Q000378:metabolism; D019976:Cloning, Organism; D019175:DNA Methylation / Q000187:drug effects; D047108:Embryonic Development / Q000187:drug effects* / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D044127:Epigenesis, Genetic / Q000187:drug effects; D005260:Female; D005347:Fibroblasts / Q000187:drug effects / Q000502:physiology; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D056572:Histone Deacetylase Inhibitors; D006657:Histones / Q000378:metabolism; D006877:Hydroxamic Acids / Q000494:pharmacology*; D008297:Male; D053652:Nuclear Transfer Techniques / Q000662:veterinary*; D014162:Transfection / Q000662:veterinary
D004791:Enzyme Inhibitors; D056572:Histone Deacetylase Inhibitors; D006657:Histones; D006877:Hydroxamic Acids; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C012589:trichostatin A