Construction And Characterization Of Nanog Reporter System In Mouse Embryonic Stem Cells Based On The High Efficient BAC Homologous Recombination

Owning to their self-renewal and multipotential differentiation abilities, embryonic stem (ES) cells have been became the first candidate for cell-based transplantation. In addition to the study on conditionally differentiation of the ES cells, the dedifferentiation of body cells to replastic its multipotence also become a bot topic. Therefore, the elucidation of ES cells' molecular mechanism to maintain its multipotential abilty is the foundation of the whole study.Previous studies have shown that there were several signal pathways including LIF/gp130/STAT3, Wnt, BMPs/Smads, PI3K etc, and several transcription factors such as OCT4,SOX2,Nanog involved in the maintainace of multipotence of ES cells. Nanog is a novel transcription factor specifically expressed in early embryonic cells and embryonic stem cells. It has been shown to play essential role in the maintenance of pluripotency in ES cells and inner cell mass (ICM) independent of LIF/Stat3 and BMPs/Smads pathways. There exists a scheduling relation among OCT4, SOX2 and Nanog in early embryonic cells and ES cells. The expression of Nanog was very sensitive to ES cells differentiation, suggesting Nanog to be one of the best markers to indicate the status of ES cells.On the other hand, homologous recombination technique based on bacterial artificial chromosome (BAC) is one of the useful techniques to construct gene targeting vectors. This technique avoids the DNA mutation and size limitation from PCR. We directly retrieve the target gene from BAC with two homo-sequences without considering enzyme sites.In this study, based on BAC homologous recombination technique, an efficient method has been developed to construct EGFP reprotor system which has been dirven by Nanog promoter. Nanog promoter was retrieved directly from the BAC, and then the reporter gene was knocked-in after it. The positive cell line R5 was picked out after the final pL253-Nanog-EGFP construct were induced into mES cells by electroporation. The new mES cell line R5 could proliferate in vitro for a long time. The new mES cell line has been detected to show features similar to their parental mES cells using the EB form and RT-PCR. The expression of EGFP can be detected in the reporter system indicating the right expression pattern of Nanog.This reporter system could effectively detect the expression level of Nanog through EGFP indicating the differentiation status of mES cells. Our study may serve as a good experimental system to study mES cells self-renewal and differentiation, facilitating the study on culture system of mES cells. We also provided an ES cell line to investigate the expression and regulation of Nanog and other factor related in mES cells, and the p1253-Nanog-EGFP construct is useful in the study of reprogramming.