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

Production of cullin 4B knockout mice and characterization of cullin 4B knockout ES cells

簡大鈞(Ta-Chiun Chien)

指導教授 ： 林淑華

國立臺灣大學/醫學院/醫學檢驗暨生物技術學研究所/碩士(2006年)

https://doi.org/10.6342/NTU.2006.00814

摘要

Cul4B屬於cullin家族，在脾臟、睪丸、卵巢及週邊血液細胞中均有多量表現，而小鼠胚胎在發育初期的8細胞時期即可偵測到Cul4B的表現。Cullin是組成cullin-ring ubiquitin ligases (CRL)的主要元件之一，而CRL可以調節細胞內許多不同種類蛋白質的分解。近年來，cullin家族中的其他蛋白與細胞週期及信息傳遞相關蛋白質的分解具相關性，但Cul4B的生理功能為何仍有待釐清。
為此，本實驗著手進行小鼠Cul4B基因剔除小鼠及條件式基因剔除小鼠的製造。目前已得到條件式Cul4B基因剔除以及Cul4B基因剔除的小鼠胚胎幹細胞株與嵌合鼠。RT-PCR與西方墨點法皆證實Cul4B在野生型的胚胎幹細胞株中有表現，在條件式基因剔除幹細胞株中表現量降低，而在基因剔除幹細胞株中無表現。經歷長期的配種，條件式基因剔除及基因剔除的嵌合鼠仍無法將標定後的Cul4B基因經由性腺傳衍到下一代。
為確認Cul4B表現量的降低對小鼠胚胎幹細胞功能的影響，本實驗針對野生型、條件式基因剔除以及基因剔除幹細胞株的細胞複製、分化能力以及幹細胞多能性進行分析。在細胞複製方面，Cul4B的缺乏並不影響胚胎幹細胞的生長速率，而流式細胞儀的分析結果顯示Cul4B表現量減少將使在G0/G1及G2/M時期的細胞比例上升。在幹細胞的分化能力方面，本實驗使用活體分化的方式，將幹細胞打入裸鼠皮下以誘導畸胎瘤的產生，染色結果顯示這三株細胞皆具有分化為三個胚層的能力。在幹細胞的多能性方面，實驗結果顯示小鼠胚胎幹細胞中的多能性標記如OCT4、AP皆不受Cul4B表現量降低之影響。此結果顯示Cul4B的缺乏有可能影響小鼠胚胎幹細胞的細胞週期。

關鍵字

Cullin 4B ； 基因剔除鼠

並列摘要

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.

並列關鍵字

Cullin 4B ； knockout mice

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