A Comparison of m-Carboxycinnamic Acid Bishydroxamide with Trichostatin A as Histone Deacetylase Inhibitor on the Developmental Competence of Somatic Cell Nuclear Transfer Porcine Embryos

서지정보

ㆍ발행기관 : 경상대학교 농업생명과학연구원 ㆍ수록지정보 : 농업생명과학연구 / 48권 / 6호
ㆍ저자명 : Mi-Ran Lee, Sang-Hoon Park, Tae-Suk Kim, Sang-Ki Baek, Sang-Jin Jin, Yeoung-Gyu Ko, Hwan-Hwoo Sung, Ho-Baek Yoon, Jin-Wook Kim, Joon-Hee Lee

목차

I. Introduction
II. Materials and Methods
2.1 Oocyte collection and in vitro maturation (IVM)
2.2 In vitro fertilization (IVF)
2.3 Parthenogenetic activation
2.4 Preparation of donor cells for SCNT
2.5 Somatic cell nuclear transfer (SCNT)
2.6 Treatment with CBHA and TSA followingactivation
2.7 Immunofluorescence staining for histone acetylation
2.8 Reverse transcription-polymerase chain reaction(RT-PCR)
2.9 Apoptosis assay of SCNT embryos
2.10 Statistical analysis
III. Results
3.1 Determination of the optimal concentration ofexposure to CBHA as a histone deacetylaseinhibitor
3.2 Determination of the optimal concentration ofexposure to TSA as a histone deacetylase inhibitor
3.3 Effects of CBHA and TSA treatment on in vitrodevelopmental competence of porcine SCNTembryos
3.4 Pluripotent genes expression of SCNT embryos atthe blastocyst stage following TSA and CBHAtreatment
3.5 Status of histone acetylation in porcine SCNTembryos following treatment of TSA and CBHA
3.6 Cell viability in porcine SCNT blastocystsfollowing treatment of TSA and CBHA
IV. Discussion
V. Acknowlegements
» Literature cited

영어 초록

The cloning efficiency is extremely low despite successful somatic cell nuclear transfer (SCNT) method producing cloned animals in several mammals. In general, faulty epigenetic modifications underlying the incomplete reprogramming of donor cell nuclei after SCNT mainly results in low cloning efficiency. The nuclear reprogramming process involves epigenetic modifications, such as DNA demethylation and histone acetylation, which may be an important factor in enhancing the cloning efficiency. Recently, the histone deacetylase inhibitors (HDACi), such as trichosatin A (TSA) and m-carboxycinnamic acid bishydroxamide (CBHA), to increase histone acetylation have been used to improve the developmental competence of SCNT embryos. Therefore, we compared the effects of TSA with CBHA on the in vitro developmental competence and pluripotency-related gene expression (Oct4, Nanog and Sox2) in porcine cloned blastocysts and histone acetylation pattern (H3K9ac). The porcine cloned embryos were treated with a 50nM concentration of TSA and 100μM concentration of CBHA during the in vitro early culture (10h) after cell fusion and then were assessed to cleavage rate, development to the blastocyst stage and pluripotency-related gene expression in NT blastocyst also, level of histone acetylation in zygote, 2cell, 4cell stage. As results, Although NT, TSA and CBHA treated NT embryos were not different between all groups for cleavage rates, the developmental competence to the blastocyst stage was significantly increased in CBHA treated embryos (22.7%) compared to that of normal NT and TSA treated NT embryos (8.1% and 15.4%)(p<0.05). In addition, all of pluripotent transcription factors (Oct4, Nanog and Sox2) were expressed in the CBHA treated NT embryos, however, Sox2 and Oct4 were expressed in TSA treated NT embryos and expression pattern of CBHA treated NT embryos is particularly similar to that of IVF embryos. Also, CBHA treated NT embryos were increased in level of histone acetylation (H3K9ac) at the zygote, 2-cell, 4-cell stage compared to those of NT and TSA treated NT embryos. In conclusion, the treatment of CBHA as a histone deacetylase inhibitor significantly increased the developmental competence of porcine NT embryos and pluripotency-related gene expressions(Oct4, Nanog and Sox2) in NT blastocysts and level of histone acetylation (H3K9ac).

참고 자료

없음