以 MultiGateway 系統以及 BAC 構築載體並建立 sox-1 knock-in及轉基因之人類胚幹細胞株

Abstract

The injury of nervous system leads to serious damage, and still it is difficult to repair the damaged nervous system because of the lack of neural stem cell in adult . The embryonic stem cells (ES cells) have the characteristic of pluripotence and self-renewal , become one of the potentially suitable materials to repair the hurt adult tissue. In the mouse model, Sex determining region Y-box 1 (Sox-1) is one of the earliest neuronal specific cell marker, and the repair efficiency of sox-1 positive neural stem cells differentiated from ES cells in vitro has already been confirmed. The purpose of my experiment is to establish the sox-1 knock-in and transgene human ES cell line. There is remarkable difference of cell specific markers between mouse and human neural stem cells . Previous research suggests that one of the earliest human rosette neural stem cells markers is paired box protein 6 (Pax-6) and at later stage sox-1 express , but the distinguishing features between this two stage are still not really clear now. The sox-1 knock-in and transgene human ES cell line can help us to examine the differentiation potential and specific quality of sox-1+ cell stage of human neural stem cell. In my experience I use two systems to construct the knock-in and transgene vector . The three-fragment multiGateway system can put two homologous arm and the reporter gene needed of knock-in vector together only with two steps site-specific recombination without considering restriction enzyme cutting sites . I use another system to construct sox-1 transgene vector, the bacterial artificial chromosome (BAC) . I choose a human BAC clone that contain sox-1 and it’s upstream and downstream sequence that may contain the sox-1 promoter and enhancer region , then we can construct the transgene vector without finding the exactly promoter region. I use a special E.coli strain EL350 that have the ability of short homologous region recombination. We can use this short homologous region recombination system to replace sox-1 region of BAC with fluorescence protein reporter gene . Finally we can transfect these two vectors into human ES cells and establish the sox-1 knock-in and transgene human ES cell line then identify it’s characters, differentiation potential and nervous system repair ability.