Study On Interspecies Somatic Cell Nuclear Transplantation Between Mouse And Pig

With the increasingly study on somatic cell nuclear transplantation (SCNT) in mammals, the interspecies somatic cell nuclear transplantation (iSCNT) has been paid more and more attention and the progresses have been made one by one in the field. Large number of studies indicated that incomplete or inappropriate epigenetic reprogramming of donor nuclei was likely to be the main cause of failure in nuclear transfer. The iSCNT may become an important model for understanding the mechanisms on epigenetic reprogramming, nuclear-cytoplasm interaction and development of early stage embryos, conducing to the protection of indangered wild animals. This technique has also behaved wide prospects for application in agriculture, medical science and related areas. In this study, mouse somatic cells have been used as the donor cells and the porcine in vitro maturation (IVM) oocyte as the recipient cells to conduct iSCNT. The resulting iSCNT embryos were cultured in5%CO2and saturated humidity CO2incubator. The purpose of this study was to research the effects of suitable conditions for donor cells or recipient oocytes on the development of mouse-pig iSCNT embryos, and the main factors impacting on iSCNT operating, so as to establish a model of in vitro produced iSCNT embryos and enable it possible to further study on reprogramming&nuclear-cytoplasmic interaction. The M16medium and37℃were commonly used in the culture of mouse embryos, while NCSU-23medium and38.5℃for porcine. The iSCNT embryos were cultured in37℃、38.5℃respectively, then cultured in NCSU-23medium、M16respectively, to study which vitro culture systems played more important part in the potentiality of the development of iSCNT embryos. According to the above results, cultured the iSCNT embryos in the optimize condition, to study the influencing factors of iSCNT furtherly, including the source of nuclear donor cells, different passage of nuclear donor cells, effect of CB addition into manipulating medium, and in vitro maturation culture time of pcrcine oocytes, et al.The results are as follows:1. Different vulva color superovulated mice were divided into two groups, vulva pink and white groups, comparing superovulation effect between the two groups. The results showed that mice with pink vulva group(81.25%) can achieve better superovulation(60.0%).2. The iSCNT embryos and mouse embryos at2-cell stage were cultured in37℃, M16medium, so as to research the adaptability of iSCNT embryos in dornor or recipient medium. Then observed the developmental potential of the two groups in mouse embryos conventional culture condition in1,2and7d. The results showed that iSCNT embryos developed lower than mouse embryonic development significantly (P<0.05), the16-cell embyo and blastula developmental rate was15.1%vs80.9%and0.0%vs27.7%(P<0.05)3. The iSCNT embryos and porcine IVF embryos were cultured in38.5℃, NCSU-23medium, so as to research the adaptability of iSCNT embryos in dornor or recipient medium.Then observed the developmental potential of the two groups in mouse embryos conventional culture condition in1,2and7d. The results showed that iSCNT embryos developed lower than mouse embryonic development significantly, the16-cell embyo and blastula developmental rate of iSCNT embryos(9.6%and0.0%), significantly lower than the porcine IVF embryos (30.6%�'�17.3%).4. NCSU-23medium was used as the iSCNT embryo culture medium, then embryos were placed in38.5℃and37℃respectively,. The effect of different culture temperatures on the potentiality of development iSCNT embryos in1,2and7d was observed. The results showed that the cleavage rate of the two groups were not significant deviation, but the iSCNT embryo development was higher in38.5℃(8-cell rate and16-cell rate was59%and13.1%) than in37℃(8-cell rate and16-cell rate was29.7%and4.7%).It is also indicated that38.5℃was a better temperature for iSCNT embryos in vitro culture.5.38.5℃was used as the mouse-porcine iSCNT embryo culture temperature, then embryos were placed in NCSU-23medium and M16medium respectively. Observe the effection of different culture medium on the potentiality of development of iSCNT embryo in1,2and7d. The results showed that iSCNT embryos development higher in NCSU-23(cleavage rate and16-cell rate was71.2%and10.9%) than M16(cleavage rate and16-cell rate was51.1%and5.3%), indicated that NCSU-23was more suit for iSCNT embryos in vitro culture.6. Using the mouse fibroblast cells and cumulus cells, the mouse fibroblast cells from2nd,3rd,4th and5th passages as donor cells, then cultured the iSCNT embryos in38.5℃and NCSU-23medium. The potential development of interspecies reconstructed embryos in1,2and7d was investigated. The result showed that both different sources and2-4passages of donor cells had no effect on the development of iSCNT embryos.7. Contrasting the effect of operating fluid with or without CB on the development of iSCNT embryos, the cleavage rate and16-cell rate of the group with CB were70.6%and12.5%, while group without CB71.1%and14.8%. Results showed that operating medium with CB had no significant effect on the iSCNT embryo development.8. Contrasting the pbI extrusion rate of porcine oocytes in42,44,46and48h, the cleavage rate of iSCNT embryos showed that44h (pbl extrusion rate, cleavage rate was77.3%and76.1%) was the best IVM time for iSCNT during this study.9. In order to identify the survival rate of iSCNT embryos,16-cell embryos were stained by FDA. The results showed that the iSCNT embryos after stained appeared green fluorescence which indicated that they were live. The survival rate was100%.