转录因子RBP－JK结合蛋白KyoT基因敲除小鼠的表型分析

KyoT is a LIM domain protein that interacts with transcription factor RBP-J. We have established a gene knock-out mouse model of KyoT. This project is primarily aimed at the analysis of expression pattern of KyoT, and thereafter the possible phnotype of KyoT deficiency. We also investigated the molecular mechanism of KyoT function. The main results we have achieved include:(1)The expression pattern of KyoT mRNA in different mouse tissues and during mouse embryogenesis was determined by Northern blot, RT-PCR, as well as in situ hybridization. We found that KyoT is expressed dorminantly in mouse testis, and the mRNA level in testis increases with the aging of mouse. The expression pattern of KyoT was confirmed by immunohistochemistry after the generation of an anti-KyoT antibody.(2). The phenotype of KyoT knock-out mice was analyzed and abnormal development of testis in KyoT-deficient mouse was found. We also found that B cell differentiation was distortured in KyoT knock-out mice, suggesting that KyoT participates lymphocyte development.(3)A cDNA library was screened using the yeast two hybrid system to isolate KyoT-interacting molecules. We detected the interaction between KyoT and human tight junction protein 2 as well as polycomb prteins RING1 and hPC2, in addition to transcription factor RBP-J. These molecular interactions were confirmed both physically and functionally. We also cloned a novel molecule, KBP, and analyzed its function in transcriptional regulation.(4)We established a protein intracellular localization-oriented functional gene cloning system, the nuclear localization signal (NLS)-trapping system. We screened a mouse embryonic cDNA library using the system, and isolated many gene fragments encoding NLS. One of the molecules, the ribosome protein L6/Taxreb107, was analyzed in detail. These results have significant impacts on the understanding of regulation of transcription factor RBP-J, a key molecule in Notch signaling pathway.

用已有的纯系杂合子KyoT基因敲除小鼠(129sv背景)与野生型129sv小鼠交配得到纯系纯合子KyoT基因敲除小鼠。同时用免疫组化RNA杂交及原位杂交等方法对KyoT在胚胎发育及成年小笞橹械谋泶锝凶既返氖笨斩ㄎ弧Ｔ诖嘶∩希治龃亢献覭yoT基因敲除小鼠的表型，庖逶谟诘贸鲎家蜃覴BP-Jk结合蛋白KyoT的体内功能及在Notch-RBP-Jk途径中的作用。