The Effect of Dnmt3L and Trichostatin A Treatment in Reprogramming of Mouse Somatic Nuclei following Nuclear Transfer
Date Issued
2010
Date
2010
Author(s)
Cheng, Dai-Han
Abstract
It has been more than a decade since “Dolly” the sheep was produced via somatic cell nuclear transfer (SCNT). Successful reprogramming of the differentiated cell by SCNT has been reported for more than 15 mammalian species that resulted in the live clones. Therefore, SCNT technology has been recognized as a powerful tool to insight into various fundamental studies in cellular, developmental, and molecular biology, as well as hold great promise in applications of agriculture and regenerative medicine. However, the cloning efficiency has remained extremely low, and was believed due to incomplete reprogramming of the somatic nuclei that contributed by insufficient epigenetic modifications.
DNA methylation plays an important epigenetic modification of gene expression in early development. It has been demonstrated that DNA methyltransferase (Dnmt) activity, including Dnmt3L, interacted with histone deacetylase (HDAC), was involved in histone deacetylation, chromatin remodeling and transcription repression. On the other hand, Trichostatin A (TSA), a histone deacetylase inhibitor (HADCi) has a function of increase histone acetylation and DNA demethylation, has been demonstrated improve the efficiency of nuclear cloning in numerous of species. If knock out Dnmt3L (Dnmt3L-KO), will it affect histone acetylation or methylation of reconstructed embryos following SCNT? Any beneficial effect during reprogramming process when combine Dnmt3L-KO nuclei with TSA treatment after SCNT? Therefore, the object of this study is to determine: 1) the role of Dnmt3L during nuclear reprogramming by SCNT, and 2) the reprogramming potential of Dnmt3L-KO fibroblast cells by nuclear transfer following TSA treatment. We compare cloning efficiency by using mouse embryonic fibroblasts derived from wild type or Dnmt3L-KO mice as donor cells for SCNT and reconstructed embryos were treated with 10 nM TSA for10 h immediately after activation. We found that cloning efficiency from both wild type and Dnmt3L-KO groups were significantly increased following TSA treatment. The development rates of cloned blastocysts compared with those observed in non-treated control groups were: 32% vs. 4% in wild type, 63% vs. 8% in D3L-KO- derived embryos. In addition, we use antibodies to detect the distribution of Oct-4/Cdx2 and divided them to Grade 1 (good) to Grade 4 (bad) together with total cell number to classify quality of cloned embryos. Combination of Dnmt3L and TSA increased not only in good quality of Grade 1 embryos but also in total cell number of cloned blastocyst. Our data indicate that TSA treatment significantly increased the developmental potential of cloned embryos and there seems a synergistic effect between Dnmt3L-KO and TSA treatment during nuclear reprogramming by SCNT.
Subjects
somatic cell nuclear transfer
Dnmt3L
TSA
nuclear reprogramming
distribution of Oct4/Cdx2
Type
thesis
File(s)![Thumbnail Image]()
Loading...
Name
ntu-99-R97626011-1.pdf
Size
23.53 KB
Format
Adobe PDF
Checksum
(MD5):21fd6dc63946ff1d12098073444f7fcd