Cullin 4B基因剔除小鼠的製造與Cullin 4B基因剔除對小鼠胚胎幹細胞功能影響之研究

Abstract

Cul4B is a member of the cullin family. High level expression of Cul4B can be detected in spleen, testis, ovary and peripheral blood cell, and also can be detected in 8-cell stage mouse embryo. Cullin proteins are the key subunits of the cullin-ring ubiquitin ligases (CRL), which regulate degradation of a wide variety of proteins in the cell. In the past few years, cullins other than Cul4B are demonstrated to be associated with the degradation of proteins in cell cycle and signal transduction pathway. The physiological function of Cul4B still remains unknown. To study Cul4B, we planed to produce Cul4B knockout mice. We disrupted Cul4B gene and produced Cul4B conditional knockout and whole body(classical) knockout murine ES cell clones, and we also produced chimeric mice from these cells. By RT-PCR and Western blotting analyses, we demonstrated the Cul4B expression decreased in the conditional knockout ES cell clones and was null in the classical knockout ES cell clones. After breeding for 13 months, no Cul4B-targeted mice were derived from these chimeras. It implicates that these Cul4B-targeted ES cells may be incapable of germ-line transmission. To investigate the effect of Cul4B protein down-regulation on murine ES cells, we set a series of experiments to examine the growth rate, pluripotency, and differentiation capacity of the wild type and Cul4B-targeted ES cells. Absence of Cul4B does not change the doubling time of ES cells, but the proportion of cells in G0/G1 phase or G2/M phase is slightly raised. To determine whether these Cul4B-targeted ES cells maintained differentiation capacity, these cells were inoculated on nude mice subcutaneously. In vivo differentiation results showed that Cul4B-targeted ES cells can differentiate into all three germ layers. To determine whether the elimination of Cul4B influences ES cell pluripotency, we detected the expression of OCT4 and AP, which are ES cell pluripotency markers. Results showed that Cul4B-targeted ES cells have the same pluripotency as wild type ES cells. These results suggest that lack of Cul4B may influence cell cycle progression of murine ES cells.