Production of mice cloned embryo by development of Somatic Cell Nuclear Transfer Technique

Abstract

Embryonic stem cells can be obtained from an embryo generated through fertilization, recently it has been reported that an embryo can also be generated asexually through Somatic Cell Nuclear Transfer (SCNT). This procedure will overcome the ethical issue regarding the usage of embryo that was initially generated for reproductive purposes. The low efficiency of reproductive cloning is one of the reasons to explore the use of SCNT for biomedical purpose, specifically through nuclear transfer Embryonic Stem Cell (ntESC) production. Cloned embryos undergo DNA hypermethylation, which is thought to be one of the causes of sub-optimal growth. This study has five main objective that include the following: (1) to obtain an optimized oocyte superovulation, activation method, and enucleation using mice oocytes as a model; (2) to produce mouse cloned embryo, parthenogenetic embryo, and in vivo fertilized embryo; (3) to study the stages of pre-implantation development in vitro culture; (4) to determined the cumulus cell cycle using flow cytometry; (5) to study the development of SCNT technique by adding Scriptaid as an inhibitory enzyme HDAC (histone deacetylase). This study showed that using doses of 7.5 IU PMSG and hCG hormones can increase the oocyte cell production optimization through superovulation treatment. The most optimal method of activation is medium CZB & CB & SrCl2 (fresh medium) for six hours (method C) to activate oocyte cells until it reaches the level of activation of 97.21%. The addition of 3% sucrose able to increase the enucleation rate. The result of the research indicated that SCNT application is able to produce cloned embryos which are capable to develop to blastocyst stage (3,2%). In addition artificial activation of oocytes could produce parthenogenetic embryos which are capable to develop up to the blastocyst stage (8,6%). The results show that cumulus cells used as nuclei donors are in the G0/G1 cell cycle phase. The cumulus cells measure at 5-7 μm in diameter and accounts for 70,84% of the total isolated cumulus cell population. The enucleation process could achieve an efficiency rate of 49%, while the nuclear transfer achieves a 40,8% efficiency rate. The addition of Scriptaid successfully increase the embryonic cloning at the blastocyst stage’s success rate to 10,8%. This efficiency rate is three times higher compared to the control, which only shows a 3,2% success rate. Optimization of the superovulation treatment, enucleation, and the activation method could improve the efficiency of SCNT application to produce cloned mice embryos. Cumulus cells were used as donor nuclei has been confirmed at G0 or G1 stage of the cell cycle. Embryos cloned mice could be generated through the SCNT application. Preimplantation development of fertilized and parthenogenetic embryos still better than the cloned embryos. The addition of Scriptaid and TSA could increase the level of efficiency of up to threefold compared with controls.