臺灣博碩士論文加值系統

精子媒介基因轉殖法具有操作簡單、低成本等優點，然而將結合外源基因之精子直接進行體外受精（in vitro fertilization, IVF），卻受限於多精入卵之障礙，難以實行。運用 ICSI 技術可有效克服多精入卵問題，此外對精子細胞膜造成損傷，則會提高外源 DNA 之結合，對基因轉殖效率有所裨益。但針對精子進行膜受損處理，會降低精子活動力，影響受精能力，必須以 ICSI 確保受精作用之發生。本試驗乃研究公豬精子與 EGFP（enhanced green fluorescent protein）基因片段行共培養後，觀察公豬精子之外源基因結合位置、結合率與結合套數，再進一步評估冷凍解凍（Frozen-thawed）與 dithiothreitol（DTT）處理對精子結合外源基因之影響。隨後利用 ICSI 技術結合精子載體法，注射帶有 EGFP之精子到成熟豬卵母細胞中，觀察卵母細胞之原核形成、受精暨隨後之胚發育，評估產生表現綠色螢光豬胚之可能性。精子獲能培養 5 小時聯合卵母細胞體外成熟培養 48 小時之處理組 ICSI 後卵母細胞存活率最高（100%），顯著高於精子獲能 5 小時聯合卵母細胞體外成熟培養 44 小時之組（86.7%），而與另外 2 組無顯著差異；就致活率而言，精子獲能 1 小時聯合豬卵母細胞體外成熟培養 44 小時者為 80.1%，顯著高於（P < 0.05）精子獲能 5 小時聯合卵母細胞體外成熟培養 44 小時之組（41.6%），而與另外 2 組無顯著差異。但雄原核形成率與精子去濃縮率並不受卵母細胞成熟與精子獲能時間之影響。將精子與 FITC-dUTP 探針共培養後，於螢光顯微鏡下發現精子頭巾後區具有明顯之螢光反應；利用 real time PCR 分析精子與 EGFP 共培養後所結合之套數，以物理性之冷凍解凍處理顯著增加 EGFP 結合套數，而經化學性之二硫蘇糖醇（DTT）共培養後之精子結合力則顯著減少。利用電穿孔法轉染 EGFP 到卵丘細胞以獲得表現綠色螢光之卵丘細胞，證明此基因可以在豬細胞表現。進一步應用冷凍解凍精子進行 ICSI，卵母細胞之雄原核形成（p = 0.087）與正常受精率（p = 0.07）均有低於 DTT 共培養組之趨勢。而 ICSI 後 48 小時之卵裂率，兩組無顯著差異，且最後兩組均未獲得表現綠色螢光之豬胚。

The method of sperm-mediated gene transfer is a simple manipulation and costs low, however, direct fertilization in vitro with sperm carrying exogenous gene is difficult to achieve the transgenic animals by polyspermy. The ICSI technique could effectively overcomes the obstacle. Making damage on the sperm membrane was benefit to transgenic efficiency because of enhances the binding of exogenous gene, but the damage treatment on sperm would decrease the sperm motility and influence the fertilization ability. It must ensure normal fertilization by ICSI. The present study was to investigate the exogenous DNA-binding site, binding rate and copies in boar sperm. Further we evaluated the effect of making damage on the sperm membrane by frozen-thawed and dithiothreitol（DTT）co-incubation treatment on the exogenous gene-binding amount of sperm. Finally we used the technology of intracytoplasmic sperm injection combined with sperm vector to inject sperm bearing EGFP into the in vitro maturated porcine oocytes. The formation of pronuclear, fertilization and embryos development were observed to estimate the possibility of producing porcine embryos expressing green fluorescence. The highest survival rate (100%) of oocytes was the group of sperm capacitated within 5 hours combined with the oocytes in vitro maturated for 48 hours following ICSI. That was higher than the group of sperm capacitated within 5 hours combined with the oocytes maturated for 44 hours (86.7%) significantly, but there was no significantly difference with other 2 groups. Just for the rate of activation, the group of sperm capacitated within 1 hour combined with the oocytes maturated for 44 hour oocytes is the highest (80.1%) and higher than the group of sperm capacitated within 5 hour combined with in vitro maturated 44 hour oocytes (41.6%) significantly, but there was no significantly difference with other groups. However, the rate of male pronuclear formation and sperm decondensation were not influenced by the time of oocytes maturation and sperm capacitation. The boar sperm was co-incubated with FITC-dUTP DNA probe and observed the fluorescence on post-acrosome under microscope. The sperm bearing exogenous DNA after co-incubated with EGFP were estimated by real time PCR. The result showed that physical treatment of frozen-thawed enhanced EGFP-binding copies but chemical treatment of DTT co-incubation decreased the binding ability significantly. The EGFP fragments were electroporated into the cumulus cells to confirm that expressed normally. Further the oocytes were injected the frozen-thawed sperm that co-incubated with EGFP previously, the rate of male pronuclear formation or decondensed sperm head, normal fertilization were all lower than the group of DTT co-incubation. The rate of cleavage at 48 hour following ICSI were no significantly difference in the two group. We never obtained any porcine embryos of expressing green fluorescence.

目錄
頁次
中文摘要 I
英文摘要 III
致謝 V
表次 5
圖次 6
壹、前言 7
貳、文獻檢討 8
一、前言 8
二、基因轉殖動物產製方法 10
（一）原核顯微注射 10
（二）反轉錄病毒感染 11
（三）胚幹細胞轉殖法與體細胞複製法 12
（四）精子媒介基因轉殖 13
（五）胞質內精子顯微注射 14
三、基因轉殖動物之應用 14
（一）改善家禽畜之經濟性能 14
（二）研究基因功能及基因療法 15
（三）生物反應器 16
（四）異種器官移植 17
四、精子媒介基因轉殖 18
（一）發展歷史 18
（二）精子結合外源 DNA 機制 19
（三）精子載體法產製基因轉殖動物 20
1. 人工授精（artificial insemination, AI） 20
2. 睪丸媒介基因轉殖（testis-mediated gene transfer, TMGT） 21
3. 體外受精（in vitro fertilization, IVF） 22
4. 胞質內精子顯微注射（intracytoplasmic sperm injection, ICSI） 23
參、材料與方法 25
一、卵母細胞之來源與體外成熟 25
二、精子之製備 29
（一）精子來源 29
（二）精子之體外獲能 29
（三）精子之膜受損處理 32
三、質體來源及前處理 32
四、精子與外源基因共培養暨 DNA 萃取 35
五、卵丘細胞培養 35
六、基因組DNA（gDNA）之萃取及定量 35
七、FITC 螢光探針製備 36
八、卵丘細胞之外源基因轉殖 36
十、精子顯微注射 43
（一）顯微注射針的製作與架針步驟 43
（二）精子顯微注射之步驟 43
十一、電激活之處理 47
十二、體外培養 50
十三、卵母細胞或胚之形態觀察 50
十四、評估指標 51
（一）成熟評估 51
（二）受精評估 51
（三）發育評估 51
十五、統計分析 57
十六、試驗設計 57
（一）精子獲能時間與豬卵母細胞成熟時間對精子顯微注射後卵母細胞受精之影響 57
（二）應用 FITC 之 EGFP 探針檢測精子之外源基因結合位 57
（三）即時定量PCR分析不同膜受損處理之精子結合外源基因之數量 57
（四）對卵丘細胞進行電穿孔轉殖 EGFP 基因 58
（五）應用結合外源基因之精子進行精子顯微注射 58
肆、結果 59
一、豬精子獲能時間與卵母細胞體外成熟時間對精子顯微注射後雄原核形
　　成之影響 59
二、豬精子與外源基因結合之分析 64
（一）公豬精子與外源基因結合位 64
（二）精子膜受損處理對結合外源基因之影響 64
三、外源EGFP 在豬卵丘細胞之表現 72
四、應用膜受損處理精子對 ICSI 後受精與胚發育之影響 74
伍、討論 80
陸、結論 85
柒、參考文獻： 86

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