5’Regulatory Sequence Cloning Of Buffalo Pluripotential Transcription Factors (Oct4,Sox2, Nanog) And Their Function Analysis

In order to explore the mechanism of transcriptional regulation of the buffalo OCT4, SOX2, NANOG upstream sequence, the5’regulatory sequences of the above-mentioned three pluripotential transcriptional factors were cloned, and the EGFP reporter vectors containing regulatory sequences of different lengths were constructed. Then the transcriptional activities of different regulatory sequences were tested in the levels of early embryos and cells.1Buffalo OCT4gene5’ regulatory sequence were cloned, and four different length (-2571bp,-2389bp,-2338bp and-2191bp) according to the distribution of2A and2B in CR4were selected to construct their EGFP reporter vectors respectively. The transgenic pig embryos were produced using the various reporter vectors by ICSI. The green fluorescent proteins could be observed under fluorescence microscope in all groups after4.5d. But no fluorescence was observed after transfecting the vectors into buffalo fetal fibroblasts (BFF)48h. QRT-PCR analysis showed that the activity of-2571bp fragment was extremely significant higher than other regulatory sequences (P<0.01). There was no significant difference between-2389bp and-2338bp(P> 0.05), and the both were extremely significant higher than-2191bp (P<0.01).2The buffalo SOX2gene5’regulatory sequence was cloned, and five regulatory sequences of different lengths (-2263bp,-1816bp,-1275bp,-660bp and-407bp) were selected to construct their EGFP reporter vectors respectively. The transgenic pig embryos were produced using the various reporter vectors by ICSI._The green fluorescent proteins could be observed in all groups except p-407-EGFP group after4.5d. No fluorescence was observed in the p-407-EGFP group after transfecting the vectors into BFF48h. But there was still a little fluorescence can be observed in other groups. QRT-PCR analysis showed that the activities of different regulatory sequences in pig4.5d embryos had a extremely significant decreasing trend with the gradual reduction of the fragments (P<0.01). In BFF, the transcriptional activity differences between any two groups were extremely significant (P<0.01). The-2263bp fragment had the highest activity, followed by-660bp, then-1275bp, and finally-1816bp.3The buffalo NANOG gene5’ regulatory sequence was cloned, and four regulatory sequences of different lengths (-2263bp,-1816bp,-1275bp,-660bp and-407bp) were selected to construct their EGFP reporter vectors respectively. The transgenic buffalo embryos were produced by injecting p-1213-EGFP into fertilized egg cytoplasm, and the green fluorescent proteins were only observed in the inner cell mass of blastocyst. The transgenic pig embryos were produced using the various reporter vectors by ICSI, and the green fluorescent proteins could be observed in all groups after4.5d. But there was only a little fluorescence could be observed in all groups after tranfecting the vectors into BFF48h. QRT-PCR analysis showed that, the transcriptional activity differences between any two groups were extremely significant in pig4.5d embryos (P<0.01). The fragment of-745bp had the highest activity, followed by-425bp, then-1213bp, and finally-312bp. The activity of-425bp fragment in BFF was extremely significant higher than others (P<0.01). There was no significant difference between-1213bp and-745bp(P>0.05), and the both were extremely significant higher than-312bp fragment (P<0.01)In conclusions, the-2191bp fragment is sufficient to regulate pluripotential cell-specific expression of OCT4. The CR4plays a role in transcriptional regulatory of OCT4in pig4.5d embryos except-2389bp～-2338bp fragment. The-660bp～-407bp fragment is an integral part of the buffalo SOX2basic promoter. Potential pluripotential cell-specific and non-pluripotential cell-specific enhancer elements are distributed in the upstream of-660bp. In buffalo blastocyst, the-1213bp fragment can mediate specific expression of buffalo NANOG gene in inner cell mass. And there are also potential non-pluripotential cell-specific regulatory elements distributed in-1213bp fragment.