Abstract:

To investigate the expression regulation mechanism of NANOG gene in swamp Buffalo, the 5′ regulatory region of NANOG gene was cloned and analyzed. Four deletion mutants fragments -1 213, -745, -425 and -312 bp were designed and constructed as EGFP reporter vectors respectively. Using the reporter vectors, the transgenic buffalo and pig embryos were produced by micro-injection method. The result showed that EGFP could only be observed in inner cells mass(ICM). In the 4.5 d pig embryos, the EGFP could be observed in all reporter vectors. After tranfecting the four reporter plasmids into buffalo fetal fibroblast about 48 h, fluorescence could be observed in a small number of cells in all groups. QRT-PCR analysis showed that the differences of the transcriptional activity from each other were highly significant in pig 4.5 d embryo cells, the -745 bp fragment had the highest activity, followed by -425 bp, then -1 213 bp and finally -312 bp. In buffalo fetal fibroblasts, the activity of -425 bp fragment was significantly higher than that of the others, there was no significant difference between -1 213 and -745 bp, and the both were significantly higher than that of -312 bp fragment. These results indicated that, in buffalo blastocyst the -1 213 bp fragment can mediate specific expression of buffalo NANOG gene in ICM. Based on the above results, we predict that there is pluripotent cell-specific inhibition element existed in -1 213--745 bp and -745--425 bp contains pluripotent cell-specific enhancer element, and -425--312 bp contains non-pluripotent cell-specific enhancer element.