The Influence Of The MicroRNA-145 And MicroRNA-483 On The Development Of The Bovine Scnt Embryos In Vitro

The SCNT technique is believed to be the most effective way to product the clone animals and transgenic animals, it has great application prospection in many fields, especially in Animal Genetics and Breeding, Animal species protection and Biological medicine. However, there is generally low efficiency of the animal production by the SCNT, it is widely believed that the abnormal reprogramming of the somatic cells in SCNT embryos would lead to the low efficiency, so it has great significance to applicate the SCNT technique widely by improving the efficiency of the reprogramming, the development rate, and the quality of the embryos. In our study, we compare the differences of the development rate between the SCNT and IVF embryos in the pre-implantation period, and investigate the gene expression differences of some reprogramming related genes, and use the microRNAs to RNA interference the gene expression to improve the development rate and quality of the bovine SCNT embryos in vitro. The results are as follows:1. Compare the differences of the development rate between the SCNT and IVF embryos in the pre-implantation period, the result shows no significant difference between the SCNT and IVF embryos in 2-cell and 4-cell stages, but the development rate of the SCNT embryos is significantly lower than that of the IVF embryos in 8-cell, morula and blastocyst stages. We further compare the differences of the expression levels of the reprogramming related genes, including the OCT4、SOX2、KLF4、IGFBP3 and IGF2, between the SCNT and IVF embryos. the results shows the gene expression levels of the OCT4、SOX2 and KLF4 are significantly lower than that of the IVF embryos in 8-cell, morula and blastocyst stages, this indicates that the differences are related to the abnormal development of the SCNT embryos; And more results show that the expression level of the gene IGFBP3 of the SCNT embryos is significantly lower than that of the IVF embryos, the expression level of the gene IGF2 of the SCNT embryos is significantly higher than that of the IVF embryos, and this indicates that the differences are related to the abnormal development of the SCNT embryos, furthermore, the overgrowth of the clone animals.2. The microRNAs could specifically down regulate the target genes, and the microRNAs inhibitor could specifically down regulate the homologous microRNA. When the 40μM microRNA-145 inhibitor is injected into the bovine SCNT embryos, the result shows that the development rate of the bovine SCNT embryos is significantly improved in 8-cell, morula and blastocyst stages. Furthermore, the expression levels of the gene OCT4、SOX2 and KLF4, which are target genes of the microRNA-145, are increased correspondingly. This indicates that the microRNA-145 inhibitor could increase the expression levels of the gene OCT4、SOX2 and KLF4, and improve the development of the bovine SCNT embryos in vitro.3. The IGFBP3 could inhibit the biological function of the IGF2 by specifically binding to the IGF2, and the IGFBP3 is the target gene of the microRNA-483. When the 100μM microRNA-483 inhibitor is injected into the bovine SCNT embryos, the result shows that the development rate of the bovine SCNT embryos is significantly improved in 8-cell, morula and blastocyst stages. Furthermore, the expression levels of the gene IGFBP3 is significantly increased, and the expression levels of the IGF2 is significantly decreased correspondingly. This indicates that the microRNA-483 inhibitor could increase the expression levels of the IGFBP3 and the increased IGFBP3 could bind to more IGF2, thus it makes the expression level of the IGF2 decreased, and corrects the abnormal embryo development which is caused by the abnormal reprogramming.