Transcriptome Analysis And In Vitro Culture Of ICM Derived From Bovine Blastocyst

Bovine embryonic stem(ES) cells have potential application in livestock industry. But no validated bovine ES cell lines has been generated up to now,this may primarily be due to that the mechanism maintaining stemness and self renewal of bovine ES cells is still unclear. With the rapid development of the next-generation sequencing technology such as Illumina, 454 and SOLID platform, RNA-seq has become a new kind of important method to study gene expression and transcriptome analysis. This study do the screening of differentially expressed genes between bovine ICM and TE using the next-generation sequencing, and culture the ICM colonies in-vitro. It was designed to investigate the mechanism maintaining stemness and self renewal and the pluripotency-related markers of bovine ES cells so as to provide a basis for optimizing culture conditions of bovine ES cells and promoting its research progress.The results were as follows:Experiment 1: screening of differentially expressed genes between bovine ICMand TE using the next-generation sequencing technologyThe ICM and TE were isolated from bovine IVF blastocysts by magnetic activated cell sorting.Then differentially expressed genes between bovine ICM and TE were screened by next-generation sequencing, and validated using q RT-PCR. ICM and TE were separated into two groups( three replicates of each group). There were a total of 207 genes that were differentially expressed significantly between ICM and TE, with 159 genes upregulated in the ICM and 48 genes downregulated in the ICM( Padj≤0.05 and |log2Ratio|≥1). In addition, Genes differentially expressed were enriched in 14 GO terms related to Biological Process(Pvalue≤0.05),and 12 pathways involved in lineage commitment and differentiation as well as those involved in maintenance of stemness and self renewal were significantly enriched(Pvalue≤0.05).Experiment 2: In-vitro culture of ICM clony derived from bovine blastocystsThe inner cell mass(ICM) were separated from bovine IVF blastocysts, seeded on mouse embryonic fibroblasts cultured in 2i/LIF medium and then passaged mechanically. The pluripotent markers of the ICM-derived colonies were detected at passage1 and passage10 by immunostaining. Additionally, the ICM and trophectoderm(TE) isolated from bovine blastocysts by magnetic activated cell sorting were used for q RT-PCR to validate the differences in gene expression between ICM and TE. We have derived ICM colonies from bovine IVF blastocysts and cultured them to passage 10,which displayed typical stem cell morphology and expressed specific markers such as OCT4, SOX2, NANOG, SSEA1, SSEA4 and TRA-1-60. Moreover, the results of q RT-PCR showed that SOX2 were differentially expressed most significantly between ICM and TE. In conclusion, 2i/LIF culture medium is helpful for the culture of the ICM-derived colonies from bovine IVF blastocysts,and SOX2 is more likely to be a candidate pluripotency-related marker gene of bovine ICM-derived colonies.