Effects Of G418 Treatment Of Donor Cells On The In Vitro Developmental Efficiency Of Cloned Porcine Embryos

Somatic cell nuclear transfer(SCNT) technology has been widely used in animal science, life science and medical science. It is the most commonly used method for production of genetically modified(GM) animals. During the creation of GM animals by SCNT, G418 is often used for selecting GM donor cells. So far the effect of G418 selection of donor cells on the developmental potency of cloned GM embryos is still unknown. To explore the influence of G418 selection of donor cells on the survival efficiency of SCNT embryos, and thereby provide scientific information for optimizing the SCNT-based transgenic animal generation procedure, in the present study, porcine nuclear donor cells were treated by G418. Expression levels of antioxidation and apoptosis-related genes were detected by quantitative RT-PCR. The DNA methylation level of genomic repeat sequences were analyzed using bisulfites sequencing analysis, and the developmental rate of SCNT embryos cloned from G418-treated donor cells were investigated in this study.The results are as follows:(1) G418 treatment significantly inhibited the proliferation of porcine nuclear donor cells and leaded to cell death, changed the morphologies of the living cell, and significantly increased the rate of the G0/G1 phase cells.(2) G418 treatment of donor cells caused significant(P<0.05) changes in expression level of genes related to antioxidation and apoptosis genes.(3) G418 treatment of donor cells significantly changed the expression of Dnmt1 and Dnmt3 a, but did not significantly(P>0.05) alter the DNA methylation degree of LINE-1, microsatellites, XIST and Bax.(4) SCNT embryos cloned from G418-treated donor cells exhibited a significantly(P<0.05) lower in vitro developmental competence than the control group.(5) The blastocyst rate of control embryos was significantly(P<0.05) lower than that of SCNT embryos cloned form donor cells received with G418 treatment followed by recovery culture.These results suggest that G418 treatment has toxic effects on donor cells, it will decrease the in vitro developmental capability of cloned embryos. However, the recovery culture following G418 treatment can eliminate the toxicity from donor cells and enhance the viability of the SCNT embryos. The reason may be that G418 treatment can select donor cells with higher apoptotic resistance and thus promote the SCNT embryo development.