鄭登貴臺灣大學：畜產學研究所蔡佩均Tsai, Pei-ChunPei-ChunTsai2007-11-292018-06-292007-11-292018-06-292004http://ntur.lib.ntu.edu.tw//handle/246246/63607本試驗旨在研究小鼠鋅指蛋白-1基因 (mouse zinc finger gene-1, mZFG-1) 在雄性可能扮演之生理功能。鑑於前人的研究證明短暫阻斷 mZFG-1S 之表現，即可嚴重影響小鼠胚之埋殖前發育，因而增加該基因調控機制相關研究之困難度；本研究乃先針對 mZFG-1L cDNA 全長設計一長度為 926bp 之反義去氧核醣核酸序列 (antisensesequence)，並將之構築於接受蛻皮素 (ecdysone) 誘導調控基因表現之載體中，進一步將此表現載體及調節載體 (regulator vector) 截切後，經純化隨即進行小鼠原核顯微注射 (pronuclear microinjection)，從而產製出可經由蛻皮素誘導該轉殖基因表現之雙轉基因小鼠，試驗結果證明共出生 66 隻仔小鼠，經應用聚合酶鏈鎖反應 (polymerase chain reaction, PCR) 及南方吸漬法 (Southern blot analysis) 皆證實有四隻係帶有雙轉基因者；此等雙轉基因小鼠經篩選及繁殖後，對施打誘導劑 ponasterone A 之成年雄小鼠進行 RNA、蛋白質萃取及冷凍切片，結果顯示經誘導表現反義核苷酸者之睪丸中，mZFG-1L 基因表現量的降低會造成生精細管中精子數目減少，此結果顯示 mZFG-1 鋅指蛋白在成年雄小鼠的生精 (spermatogenesis) 過程中可能扮演一重要的角色。 進一步試驗經由RNA干擾 (RNA interference, RNAi)、二維膠體電泳 (two-dimensional polyacryamide gel electrophoresis, 2D-PAGE) 及質譜分析策略，探討 mZFG-1 表現量遭受 siRNA 干擾後，對於睪丸賽透力氏細胞 (Sertoli cell) 及萊迪吉氏細胞 (Leydig cell) 中下游基因表現之影響；試驗結果發現在賽透力氏細胞株中，兩個和生殖內分泌及生精作用有關之蛋白 Crisp-1 (Cysteine-rich secretory protein-1) 及 NPY (Neuropeptide Y) 會受 mZFG-1 的正向調控，即 mZFG-1 的表現量降低時兩者的表現量亦隨之下降，由於已知 Crisp-1及 NPY 為維持雄性正常生殖作用所不可或缺者；故初步試驗結果顯示，mZFG-1在睪丸中可能透過直接或間接的調控 Crisp-1 及 NPY 的表現而維持雄性小鼠正常的生精作用；此蛋白質體分析得到的結果與轉基因鼠發生的現象頗相契合，惟詳細的機制仍待進一步的探討。The purpose of this study was to clarify the molecular mechanisms of mouse zinc finger protein-1 (mZFG-1) gene potentially involved in the regulation of spermatogenesis within the mouse testis. To meet this purpose, an initial attempt was made to generate transgenic (Tg) mice characterized by conditional knock-down of the mZFG-1 gene. Of these studies, a transgene named anti-mZFG-1L/pIND/V5-His-TOPO, containing antisense transcript against to nucleotide sequences complementary to the long form of mZFG-1 cDNA, was constructed. Generation of Tg mice was conducted by co-injection transgenes of anti-mZFG-1L/pIND/V5-His-TOPO and pVgRXR, into the pronucleus of newly fertilized eggs. These have resulted in 10 out of 66 newborn mice to be transgenic and four out of these 10 Tg mice were confirmed, as evidences shown by PCR and Southern-blot analyses, to be harboring both of the transgenes said above. Each of the four double-transgenic founder mice appeared to be fertile with 16.7 ~ 100% of their transgenes being germline-transmitted to the F1 progenies obtained. Evidences from RT-PCR analyses further confirmed that, in comparison with those of the control mice, much less of endogenous mZFG-1 mRNA was found in the double-transgenic founder mice when they had been subjected to the ponasterone-A induction. While no gross anomalies was found in any somatic tissues from those ponasterone-A induced double-transgenic mice, much less number of mature spermatozoa were found in testis-specimens from the double-transgenic mice when comparison was made to those of control mice after ponasterone-A induction. Moreover, the effect of mZFG-1 on spermatogenesis at molecular level was further verified using mouse Sertoli cell lines (TM4 cell lines) with the specific small interferon RNA (siRNA) strategy for knock-down the expression of endogenous mZFG-1 and the consequent expression profiles of its down-stream genes were investigated. Based on results from RT-PCR, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry (MS) analyses appeared that both Crisp-1 (Cysteine-rich secretory protein-1) and NPY (Neuropeptide Y) genes were down-regulated within Sertoli cells when they had been transfected with siRNA designed against to mZFG-1 gene. Conclusions came to these present studies were that appropriate expression of mZFG-1 gene do play its physiological significances for ensuring normal spermatogenesis occurred within the testes and the potential effect of mZFG-1 gene on spermatogenesis may be mediated by modulation the expression of both Crisp-1 and NPY genes.目 錄 頁次 目錄………………………………………………………………………… I 表次………………………………………………………………………… III 圖次………………………………………………………………………… IV 摘要………………………………………………………………………… 1 前言………………………………………………………………………… 2 文獻檢討 壹、研究基因功能的方法……………………………………………… 3 貳、鋅指蛋白簡介……………………………………………………… 13參、睾丸的發育………………………………………………………… 17 肆、小鼠鋅指蛋白基因-1的研究背景………………………………… 28 材料與方法 一、選用的模式動物…………………………………………………… 30 二、轉染所用之細胞株及其培養條件………………………………… 30 三、組織包埋、冷凍切片及免疫組織化學染色法分析……………… 31 四、基因的構築………………………………………………………… 33 五、反義mZFG-1轉基因小鼠的產製及分析………………………… 46 六、降解mZFG-1基因於TM4及TM3細胞株之分析……………… 54 結果與討論 一、小鼠胚胎發育期間及成體睪丸中mZFG-1蛋白質的表現模式… 59 二、構築誘導性外源反義基因之載體………………………………… 62 三、雙轉基因鼠之產製及外源基因的嵌插分析結果………………… 65 頁次 四、誘導雙轉基因鼠表現反義基因的表現及分析…………………… 68 五、構築降解 mZFG-1 之 si-RNA 載體…………………………….. 68 六、轉染shRNA載體於賽透力氏 (TM4) 及萊迪吉氏細胞株 (TM3) 並分析 mZFG-1 基因的表現…………………………… 71 七、TM4及TM3細胞於mZFG-1被降解後其蛋白質表現模式…….. 74 結論…………………………………………………………………………. 81 參考文獻…………………………………………………………………… .82 英文摘要……………………………………………………………………. 99 小傳………………………………………………………………………… 101 Index of Tables 頁次 Table 1. Oligo primers used for all analysis………………………………… 39 Table 2. Germline transmission of anti-mZFG-1L/pIND/V5-His- TOPO transgene in transgenic founder after breeding with that of nontransgenic mice………………………………………… 66 Table 3. Germline transmission of pVgRXR transgene in transgenic founder after breeding with that of nontransgenic mice…………… 66 Table 4. Protein affected by mZFG-1 knock down were identified in TM4 cell line……………………………………………………………… 77 Index of Figures 頁次 Figure 1. Schematic representation of the cellular components of the gonad and their fate during early testicular development………… 24 Figure 2. Outline of the interaction of the molecular players involved in early testicular development…………………………………….. 25 Figure 3. Ecdysone-regulated transgene expression………………………. 34 Figure 4. Blocking nucleic targets by antisense oligonucleotides…………. 38 Figure 5. Comments for anti-mZFG-1L/ pIND/V5-His-TOPO construct strategy………………………………………………………….. 40 Figure 6. Vector-expressed shRNA function to knock down mRNA expression………………………………………………………. 42 Figure 7. Hairpin siRNA template design for knockdown mZFG-1……… 44 Figure 8. Comments for si-mZFG-1 / U6-EGFP-C1 construct strategy….. 45 Figure 9. Expressional patterns of mZFG-1L were detected in post-implantation stages by immunohistochemistry…………… 60 頁次 Figure 10. Localization of mZFG-1L analyses in mouse testis by immunohistochemistry………………………………………. 61 Figure 11. Primer sets and restriction maps of anti-mZFG-1L/pIND/V5- His-TOPO and pVgRXR transgenes………………………… 63 Figure 12. (a) Screen anti-mZFG-1L/pIND/V5-His-TOPO E.coli strain with PCR analysis by primer Ecdysone F and ZF255. No.4, No.16 and No.22 are positive clone.(b) Double transgenic mice harboring anti-mZFG-1L/pIND V5-His-Topo & pVgRXR confirmed by PCR…………………………………………… 64 Figure 13. Mouse genomic DNA Southern-blot hybridization result…… 67 Figure 14. RT-PCR (a) and Western blottong (b) analysis of mZFG-1L transcript expressed in in testes anti-mZFG-1L/pIND & pVgRXR double-transgenic mice after treatment with sesame oil (control) and 2 mg ponasterone A (ponA)……… 69 Figure 15. Comparing testicular histology of normal and induced anti- mZFG-1L / pIND / His-TOPO & pVgRXR double-transgenic mice………………………………………………………….. 70 頁次 Figure 16. Expression of mZFG-1L was timely down-regulated in TM4 (Sertoli cell)…………………………………………………. 72 Figure 17. Expression of mZFG-1L was timely down-regulated in TM3 (Leydig cell)………………………………………………… 73 Figure 18. Protein changes due to mZFG-1L decrease in TM4 (Sertoli cell) line by taking use of small interference RNA… 75 Figure 19. Comparison of protein disruption in TM3 (Leydig cell) transfected with or without mZFG-1L siRNA……………… 76 Figure 20. Crisp-1 and NPY transcripts were down-regulated in TM4 (Sertoli cell) that the mZFG-1L was knocked by RNA interference………………………………………………… 781458212 bytesapplication/pdfen-US鋅指蛋白小鼠生精作用mousespermatogenesiszinc fingerthesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/63607/1/ntu-93-R90626002-1.pdf