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研究生:林芳羽
研究生(外文):Fang-Yu Lin
論文名稱:利用基因剔除鼠分析Supt4h在大腦的作用
論文名稱(外文):Using conditional knockout mice to analyze Supt4h function in the brain
指導教授:鄭子豪顏裕庭顏裕庭引用關係
指導教授(外文):Tzu-Hao ChengYu-Tin Yan
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生化暨分子生物研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2016
畢業學年度:104
語文別:英文
論文頁數:46
中文關鍵詞:Supt4h基因剔除鼠表型分析
外文關鍵詞:Supt4hConditional knockout micePhenotypic analysis
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Supt4h,已知為一個在真核生物演化上高度保留的轉錄延伸因子。實驗室先前利用酵母菌研究證實,當Supt4h的同源基因Spt4基因缺失時,可以選擇性減少異常擴增的CAG重複序列的表現量,進而降低polyQ蛋白聚集體的產生,但不會影響正常重複序列的基因表現。而在哺乳類細胞中,Supt4h會和Supt5h結合在一起形成異質二聚體,參與並協助RNA聚合酶II的轉錄作用。然而,Supt4h在神經系統中的功能仍屬未知。先前的研究指出全身性剔除Supt4h會造成小鼠在胚胎發育早期死亡,為了更進一步了解Supt4h在大腦中的功能,在此建立組織特異性 (tissue-specific)基因剔除小鼠 (conditional knockout mice,cKO mice)。結果顯示,在神經發育初期,發現利用Nestin基因啟動子來啟動Cre重組酶(recombinase)的表現將神經前驅細胞的Supt4h剔除掉會造成小鼠因為無法正常執行呼吸功能而在出生後死亡。同時也觀察到基因剔除小鼠在前腦有許多組織學上的缺陷,例如海馬迴(hippocampus)的發育以及大腦皮層(cortex)分層上的異常等。另一方面,在小鼠大腦成熟後期,利用Camk2a基因啟動子來啟動Cre重組酶的表現將小鼠大腦皮質層以及海馬迴的Supt4h剔除,在初步的表型觀察並沒有發現明顯異常。總結以上結果,顯示Supt4h在神經發育上扮演不可或缺的角色;一旦進入成鼠階段,大腦的神經細胞對於Supt4h的依賴性有降低的趨勢,而此現象和機制尚需進一步的分析。
Supt4h is known to be a transcription elongation factor that is highly conserved among eukaryotes. In previous studies, SPT4 was identified through genome-wide genetic screening in yeast and showed transcriptional control of genes encoding a long stretch of polyQ in a selective manner. Supt4h, the mammalian ortholog of Spt4, is associated with Supt5h to promote transcription elongation and RNA polymerase II processivity, and modulates the expression of mutant Htt allele in neuronal cells. Nevertheless, the biological function of Supt4h in the central nervous system is still unclear. To this end, two different lines of conditional knockout mice were breed in this study. We found that ablation of Supt4h in neural progenitor cells causes the postnatal lethality due to the respiratory failure. In addition, multiple defects, including agenesis of the hippocampus and the incorrect lamination of neocortex were detected. Conversely, removal of Supt4h in the adult brain, particularly in CA1 region and the superficial layers of cortex, did not have overt phenotypes based on the preliminary studies
Chinese abstract I
Abstract II
Contents III
Introduction 1
Neuronal development in mammalian brain 1
Corticogenesis in the mammalian neocortex 1
Hippocampal development in the mammalian brain 2
Regulation of the respiratory network in nervous system 3
Biochemical function of Supt4h 3
Supt4h is essential for embryogenesis 4
Materials and methods 5
Animals 5
Genotyping analysis 6
Tissue collection and protein isolation 6
Western blot analysis 7
X-gal staining 7
Immunofluorescence 8
Hematoxylin and Eosin staining 8
Nissl staining 9
Diaphragm whole-mount staining 9
Statistical analysis 9
Results 10
1. Breeding strategy of Supt4h conditional knockout and LacZ reporter lines 10
2. Supt4h broadly expresses in neurons at the embryonic and adult stages 11
3. Genetic ablation of Supt4h in neural progenitor cells results in neonatal lethality and dysfunction of respiratory networks 11
4. Genetic deletion of Supt4h in neuronal progenitors shows normal pattern of diaphragmatic innervation and neuromuscular junction formation at E18.5. 13
5. Genetic knockout of Supt4h in neuronal progenitor cells results in an abnormal pattern of respiratory center 13
6. Multiple neuronal developmental defects in Nestin-cre driven Supt4h cKO brain 14
7. Misdistribution of both progenitor and post mitotic neurons in the cortex due to the ablation of Supt4h 15
8. Differential reduction of Supt4h protein in the forebrain regions in Camk2a-Cre driven Supt4h conditional knockout mice 17
9. Inactivation of Supt4h in forebrain at adult stage caused a reduction of cell number in the superficial layers of cortex but sustains normal pattern of hippocampus 18
Discussion 19
Reference 24
Figures and tables…………………………………………………………………...28

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